hepatitis e virus in shellfish of iberian peninsula: a ... · hepatitis e virus (hev) is known as a...

Danielle de Araújo Cruz Oliveira

Hepatitis E virus in shellfish of Iberian Peninsula:

A risk for public health

Porto, setembro, 2015

.

Trabalho realizado no Laboratório de Microbiologia, Departamento de Ciências e

Biologia da Faculdade de Farmácia da Universidade do Porto, sob a orientação da

Professora Doutora Maria de São José Garcia Alexandre.

Dissertação do 2º ciclo de estudos

conducente ao grau de Mestre em Controlo

de Qualidade, apresentada à Faculdade de

Farmácia da Universidade do Porto.

i

ACKNOWLEDGMENTS

The development of this work has brought me personal capital gains and compelling

and outstanding professionals. Without the collaboration and availability of some

persons would not be possible.

First I have to thank to Professor Maria de São José Alexandre the available time and

requirement that guided this work and transmission of knowledge, by living, for her

support, understanding and friendship my most sincere thanks without saying how

much I admire her as a professional, as sympathy in person.

I also thank my lab colleagues for assistance and co-work Carolina Rebelo and Joana

Silva, really it was very satisfying our coexistence with respect and collaboration.

A big thank you to Dra. Joana, from Bromatology department and Molecular Biology

laboratory, for always receive me to take doubts. That was true lessons. You helped a

lot!

Thanks for my husband for supporting and encouraging me in achieving this master, for

your patience and love.

Thanks for my family despite being away will always be my strength and inspiration to

accomplish all the goals I set myself.

Thanks for my mother and father in law for supporting in everything.

ii

The work developed in this dissertation originated the following publication:

Mesquita, JR; Oliveira, D.; Abreu-Silva, J.; Varela, MF; Rivadula, E; Romalde, JU;

Nascimento, MSJ. 2015. Hepatitis E vírus genotype 3 in mussels (Mytilus

galloprovincialis), Spain. [Submitted to Journal of Emerging Infectious Diseases].

iii

ABSTRACT

Hepatitis E virus (HEV) is known as a leading cause of acute hepatitis linked to fecal-

oral transmission in developing countries. However in the last decade an increasing

number of autochthonous cases of HEV infection were recorded in industrialized

countries. A zoonotic transmission has been linked to these cases through undercooked

HEV contaminated pork meat intake or to direct contact. Infectious HEV has been

found in swine manure therefore, application of manure to land and subsequent runoff

to coastal water, can lead to a contamination of shellfish cultivated in this contaminated

environment. The consumption of these shellfish, usually lightly cooked or raw, could

be an important route of HEV infection.

The aim of the present study was to search for the presence of HEV RNA in shellfish

samples collected in a coastal area of Galicia, Spain, and to characterize the HEV

positive samples in order to evaluate the genetic relationship with strains already

known.

Shellfish samples (n=81) were tested for the presence of HEV RNA using three in

house real-time RT-PCR methods (Qiagen OneStep RT-PCR, Kapa Fast Universal One-

Step qRT-PCR, One Step Kapa Sybr® Fast qPCR) targeting a conserved region of the

open reading frame (ORF) 3 of HEV genome. Positive samples were further tested with

a nested RT-PCR targeting the ORF1 of HEV. The obtained amplified products (330bp)

were sequenced and submitted to phylogenetic analysis.

Six samples presented the expected amplicon that clustered with sequences classified as

HEV genotype 3 subgenotype e, and showed to be closely related to human strains.

This is the first study reporting the presence of HEV in shellfish cultivated in Iberian

Peninsula thus demonstrating a potential risk to public health.

Keywords: Hepatitis E virus, HEV, shellfish, real-time RT-PCR

iv

RESUMO

O vírus da hepatite E (HEV) é conhecido como a principal causa de hepatite aguda

associada à transmissão fecal-oral nos países em desenvolvimento. No entanto, na

última década tem sido registado nos países industrializados um número cada vez maior

de casos de infeção autóctone pelo HEV. A transmissão zoonótica tem sido associada a

estes casos pela da ingestão de carne de porco mal cozida contaminada com HEV ou

por contacto direto. O HEV já foi encontrado em excrementos de suínos, portanto, a sua

aplicação para adubação da terra e o subsequente escoamento destes dejetos para as

águas costeiras, poderia levar à contaminação de moluscos bivalves cultivados nestas

áreas. O consumo de bivalves, normalmente crus ou ligeiramente cozidos, poderia ser

uma importante via de infecção pelo HEV.

O objetivo do presente estudo foi pesquisar a presença de HEV RNA em amostras de

moluscos bivalves coletados em uma área costeira da Galiza, Espanha, e para

caracterizar as amostras positivas HEV, a fim de avaliar a relação genética com cepas já

conhecidas.

As amostras foram testadas (n = 81) foram testados quanto à presença de HEV RNA

utilizando três métodos in house de RT-PCR em tempo real (Qiagen OneStep RT-PCR,

Kapa Fast Universal One-Step qRT-PCR, a um passo One Step Kapa Sybr® Fast qPCR)

com primers ligando-se a uma sequência na região ORF 3 do genoma do HEV. As

amostras positivas foram também testadas com nested RT-PCR com primers ligando-se

à região ORF1 do HEV.

Os produtos amplificados obtidos (330pb) foram sequenciados e submetidos à análise

filogenética. Seis amostras apresentaram o fragmento amplificado esperado que

agrupado com sequências classificadas como HEV genótipo 3 e subgenótipo, e

mostrou-se intimamente relacionada com cepas humanas.

Este é o primeiro trabalho que documenta a ocorrência de HEV em bivalves cultivados

na Península Ibérica evidenciando assim um potencial risco para a saúde pública.

Palavras chave: Vírus da Hepatite E, HEV, Moluscos bivalves, RT-PCR em tempo real

v

INDEX

ACKNOWLEDMENTS ............................................................................................ i

ABSTRACT .............................................................................................................. iii

RESUMO .................................................................................................................. iv

LIST OF FIGURES ................................................................................................... vii

LIST OF TABLES .................................................................................................... viii

ABBREVIATIONS ................................................................................................... x

INTRODUCTION ..................................................................................................... 1

1.1 Hepatitis E virus: general characterization ..................................................... 2

1.1.1 Epidemiology of human HEV infection ............................................. 3

1.1.2 HEV Infection symptoms ................................................................... 5

1.1.3 Hepatitis E virus hosts and reservoirs ................................................ 6

1.2 Environmental routes of HEV Transmission .................................................. 7

1.2.1 Sewage and Animal Manure Run-off ................................................. 8

1.2.2 Surface Water ..................................................................................... 8

1.2.3 Coastal Water ..................................................................................... 9

1.3 Foodborne transmission .................................................................................. 10

1.4 Presence of HEV in shellfish .......................................................................... 10

1.5 Detection of HEV in food ............................................................................... 12

1.5.1 Real-time PCR .................................................................................... 12

1.5.2 Real-time PCR design ........................................................................ 14

1.5.3 Analytical verification ........................................................................ 19

1.6 Internal Quality Control: Controls and inhibitors of PCR assays ................... 21

1.6.1 Process Controls ................................................................................. 22

1.7 Inhibition of PCR assays ................................................................................. 22

2. OBJECTIVES ........................................................................................................ 24

3. MATERIALS AND METHODS .......................................................................... 26

3.1 Samples ........................................................................................................... 27

3.1.1 HEV and mengovirus ......................................................................... 27

3.2 Real-time RT-PCR for detection of HEV RNA ............................................. 27

3.2.1 Primers and probe ............................................................................... 28

vi

3.2.2 Qiagen OneStep RT-PCR ................................................................... 28

3.2.3 Kapa Fast Universal One-Step qRT-PCR .......................................... 29

3.2.4 One Step Kapa Sybr® Fast qPCR ....................................................... 31

3.3 Nested RT-PCR for the detection of HEV RNA ............................................ 32

3.3.1 Primers................................................................................................ 32

3.3.2 Nested RT-PCR .................................................................................. 33

3.4 Electrophoresis and purification of amplified products .................................. 34

3.4.1 Sequencing of HEV nested RT-PCR products ................................... 35

3.5 Real-time RT-PCR for mengovirus ................................................................ 35

3.5.1 Primers and probe ............................................................................... 35

3.5.2 Qiagen OneStep RT-PCR ................................................................... 36

3.5.2 Evaluation of viral mengovirus RNA extraction efficiency ............... 37

4. RESULTS AND DISCUSSION ............................................................................ 38

4.1 Detection of HEV RNA in shellfish by Qiagen qRT-PCR ............................. 39

4.1.1 Detection limit of the Qiagen qRT-PCR ............................................ 39

4.1.2 HEV RNA detection in shellfish by Qiagen qRT-PCR ..................... 41

4.2 Detection of HEV RNA in shellfish by Kapa qRT-PCR ................................ 43

4.2.1 Detection limit of Kapa qRT-PCR ..................................................... 43

4.2.2 HEV RNA detection in shellfish by Kapa qRT-PCR ......................... 45

4.3 Detection of HEV RNA in shellfish by Kapa Sybr® qPCR ............................ 48

4.3.1 Kapa Sybr® qPCR Optimization ........................................................ 48

4.3.2 Detection limit of Kapa Sybr® qPCR ................................................. 50

4.3.3 HEV RNA detection in shellfish by Kapa Sybr® qPCR .................... 52

4.4 Detection of HEV RNA by nested RT-PCR ................................................... 56

4.5 Sequencing of HEV nested RT-PCR products ............................................... 59

4.6 Evaluation of viral RNA extraction efficiency ............................................... 60

5. CONCLUSION ..................................................................................................... 64

REFERENCES .......................................................................................................... 66

vii

LIST OF FIGURES

Figure 1: Scheme showing the organization of the three viral open reading frames

(ORFs) ....................................................................................................... 2

Figure 2: Geographical distribution of prevalence of human hepatitis E and

genotypes ................................................................................................ 5

Figure 3: Real-time PCR detection using TaqMan® probes 5’-3’ polymerase and

exonuclease activity of the Taq DNA polymerase (TAQ). .................... 17

Figure 4: Real-time PCR detection using Sybr Green® ........................................... 19

Figure 5: Establishment of the Qiagen qRT-PCR Standard curve for HEV ............ 40

Figure 6: Establishment of the Kapa qRT-PCR standard curve for HEV ................ 44

Figure 7: Optimization for HEV detection by Kapa Sybr® qPCR ........................... 47

Figure 8: Melting curves of the serial dilutions with annealing temperature of

64ºC ........................................................................................................ 48

Figure 9: Amplification curves of standards stock solution Kapa Sybr® qPCR. ..... 50

Figure 10: Amplification curves of shellfish samples tested undiluted by Kapa

Sybr® qPCR. ........................................................................................... 52

Figure 11: Amplification plot of samples tested diluted by Kapa Sybr® qPCR

that were considered positive.................................................................. 54

Figure 12: Electrophoresis gel of amplified products after nested RT-PCR

shellfish samples ..................................................................................... 56

Figure 13: Phylogenetic tree based on the nucleotide sequence of the ORF1. ........ 58

Figure 14: Mengovirus amplification curves of undiluted and diluted shellfish

samples by real-Time RT-PCR ............................................................... 61

viii

LIST OF TABLES

Table 1: Primers and probe used for the detection of HEV RNA by real-time RT-

PCR. .......................................................................................................... 28

Table 2: Mastermix for detection of HEV RNA by Qiagen qRT-PCR. ................... 28

Table 3: Thermal conditions for Qiagen qRT-PCR. ................................................. 29

Table 4: Mastermix for detection of HEV RNA by Kapa qRT-PCR ....................... 30

Table 5: Thermal conditions for Kapa qRT-PCR ..................................................... 30

Table 6: Mastermix for detection of HEV RNA by Kapa Sybr® qPCR. .................. 31

Table 7: Thermal conditions tested for optimization of HEV RNA detection by

Kapa Sybr® qPCR ..................................................................................... 32

Table 8: Primers used for detection of HEV RNA by nested RT-PCR. ................... 32

Table 9: Mastermix for detection of HEV RNA by nested RT-PCR - first round ... 33

Table 10: Thermal conditions for nested RT-PCR - first round. .............................. 33

Table 11: Mastermix for detection of HEV RNA by nested RT-PCR - second

round ....................................................................................................... 34

Table 12: Thermal conditions for nested RT-PCR - second round .......................... 34

Table 13: Primers and probe used for mengovirus detection ................................... 36

Table 14: Mastermix for detection of mengovirus by Qiagen qRT-PCR ................. 37

Table 15: Thermal conditions for detection of mengovirus by Qiagen qRT-PCR ... 37

Table 16: Ct values and number of copies of HEV RNA by Qiagen qRT-PCR ...... 39

Table 17: Ct values and interpretation of results of undiluted and diluted shellfish

samples for the detection of HEV RNA by qiagen qRT-PCR ............... 42

Table 18: Ct values and number of copies of HEV RNA by Kapa qRT-PCR. ........ 43

ix

Table 19: Ct values and interpretation of undiluted shellfish samples for the

detection of HEV RNA by Kapa probe qRT-PCR ................................... 45

Table 20: Comparison of the interpretation of results of 22 shellfish samples

tested by both Qiagen and Kapa qRT-PCR .............................................. 46

Table 21: Ct values and melting curve of HEV RNA stock solution by Kapa Sybr®

qPCR .......................................................................................................................... 49

Table 22: Ct values, melt temperatures and interpretation of results of undiluted

and diluted 1:5 shellfish samples by Kapa Sybr® qPCR for the

detection of HEV RNA ........................................................................... 51

Table 23: Results of the detection of HEV RNA in shellfish samples by nested

RT-PCR ..................................................................................................... 55

Table 24: Comparison of the results by real-time RT-PCR and nested RT-PCR

assays in 33 shellfish samples ................................................................ 57

Table 25: Extraction efficiency of undiluted and diluted shellfish samples spiked with

mengovirus ................................................................................................ 60

x

ABBREVIATIONS

BSA Bovine serum albumin

C Citosin

Ct Threshold cycle

Cq Quantification cycle

DABCYL 4-((4-(dimethylamino) phenyl) azo) benzoic acid

DNA Deoxyribonucleic acid

FAM 6-car-boxyfluorescein

FRET Fluorescence resonance energy transfer

G Guanine

HEV Hepatitis E virus

HEX Hexacholoro-6-carboxyfluorescein

JOE 2,7-dimethoxy-4,5-dichloro-6-carboxy-fluorescein

mM Milimolar

MNV-1 Murine norovirus 1

nM Nanomolar

nt Nucleotide

ORF Open reading frame

PCR Polymerase chain reaction

PFU Plaque-forming units

qPCR Quantitative real-time PCR

RFU Relative fluorescence units

RNA Ribonucleic acid

RT Reverse transcription

RT-PCR Reverse transcription PCR

SPC Sample process control

Ta Annealing temperature

TAMRA 5-Carboxytetramethylrhodamine

xi

TET Tetrachloro-6-car-boxyfluorescein

Tm Melting temperature

1

1. INTRODUCTION

_______________________________________________

2

1. INTRODUCTION

1.1 Hepatitis E virus: general characterization

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide

(Sridhar et al., 2015). Originally considered to be restricted to humans, it is now clear

that HEV viruses have several animal reservoirs with complex ecology and genetic

diversity and it is responsible for epidemics and endemics of acute hepatitis in humans,

mainly through waterborne, foodborne, and zoonotic transmission routes (Yugo &

Meng, 2013).

HEV is the sole member of the family Hepeviridae (Mirazo et al., 2013). Is a small,

non-enveloped, positive sense, single-stranded RNA virus (Yugo & Meng, 2013). The

genome (Figure 1) has a size of approximately 7.2 kb and consists of three open reading

frames (ORF) (Kumar et al., 2013). These three ORFs encode a non-structural

polyprotein (ORF1), the capsid protein (ORF2) and a phosphoprotein (ORF3)

associated with signal transduction (Johne et al., 2009). The genome is capped at the 5′

end and polyadenylated at the 3′ end (Smith, 2001; Xia et al., 2008; Johne et al., 2009).

Based on whole genome sequencing, HEV has been characterized into four major

genotypes (1-4) (Kumar et al., 2013). HEV genotypes 1 and 2 are only found in humans

and account for most of the hepatitis E in the developing world where transmission

occurs by the fecal-oral route via contaminated waterways (Sridhar et al., 2015). HEV

genotypes 3 and 4 are found in swine and other animals across the world (Sridhar et al.,

2015).

Figure 1: Scheme showing the organization of the three viral open reading frames (ORFs). (Mirazo et al.,

2014).

http://vir.sgmjournals.org/content/91/3/750.long#ref-37

http://vir.sgmjournals.org/content/91/3/750.long#ref-48

3

1.1.1 Epidemiology of human HEV infection

Hepatitis E affects humans in both industrialized and developing countries worldwide.

Although HEV was first known as a leading cause of acute hepatitis linked to fecal-oral

transmission in developing countries, hepatitis E has been found to be endemic in Japan,

Australia, the United States, and Europe (Dalton et al., 2008a; Purcell & Emerson,

2008).

Industrialized countries experience sporadic and cluster cases of hepatitis E. The

seroprevalence data from industrialized countries suggests that subclinical or

unrecognized infection is common. The incidence of autochthonous hepatitis E is not

known (Dalton et al., 2008a) and few data are available on the risk factors and

contamination pathways involved in acute indigenous hepatitis E in developed countries

(Renou et al., 2008). The prevalence of HEV IgG antibodies in low-incidence

populations in industrialized countries ranges from 3% in Tokyo, Japan (Ding et al.,

2003), 3.2% in central France (Boutrouille et al., 2008), 7.3% in Catalonia, Spain (Buti

et al., 2006), 16.6% in southwest France (Mansuy et al., 2008), 16% in southwest

England (Dalton et al., 2008b), to 21.3% in US blood donors (Thomas et al., 1997).

Hepatitis E is highly endemic in the Indian subcontinent, China, Southeast and Central

Asia, the Middle East, and northern and western parts of Africa (Aggarwal & Naik,

2009, Aggarwal, 2011). In these areas, outbreaks of hepatitis E of variable sizes have

been reported. In addition, a fairly large proportion of cases with sporadic acute

hepatitis in these areas are caused by HEV infection. Water-borne transmission is the

most common route for acquisition of infection. Large outbreaks of frequently occur

due to fecal-oral transmission, usually through contamination of drinking water (Kumar

et al., 2013). It is plausible that food-borne transmission also plays a role in the

transmission of hepatitis E in these regions. However, this is difficult to prove because

of the relatively long incubation period and the consequent difficulties in attributing

disease to consumption of a particular food (Aggarwal, 2011). Both outbreaks and

sporadic cases of hepatitis E in these regions have been related to genotype 1 or 2; in

some areas, a proportion of sporadic cases have been related to genotype 4 (Aggarwal &

Naik, 2009; Aggarwal, 2011). For these countries this is an important public-health

concern because it causes large epidemics and waterborne outbreaks due to poor

http://www.sciencedirect.com/science/article/pii/S147330990870255X?np=y#bib97



http://www.sciencedirect.com/science/article/pii/S0168170211001109?np=y#bib0015




4

sanitation conditions and a hyperendemic status in the population (Teo, 2010; Mirazo et

al., 2013).

In comparison, in the developed parts of the world, e.g. western Europe, USA, Japan

and Australia, hepatitis E is uncommon. Till nearly 10 years ago, cases of hepatitis E in

these areas were all related to travel to areas where the disease is common. Acute cases

of hepatitis E were reported in travelers returning from endemic regions although

sporadic cases have also been reported in patients with no known epidemiological risk

factors (Pilar et al., 2003; Lewis et al., 2008) what can be attributed as a result of

zoonotic transmission associated with ingestion of contaminated animal meat,

especially swine, shellfish, and contact with infected animals (Teo, 2010; Meng, 2013;

Said et al., 2013). The high prevalence of HEV infection among persons with

occupational exposure to swine suggests animal-to-human transmission of this infection

(Drobeniuc et al., 2001). However, in recent years, several case reports and case-series

of locally acquired (autochthonous) hepatitis E have been published from these areas.

Autochthonous hepatitis E in these areas has differences in routes of transmission and in

clinical features than those reported from the areas where hepatitis E is hyperendemic

(Aggarwal & Naik, 2009; Pavio & Mansuy, 2010). The locally acquired HEV infection

in these areas has been found to be related to genotype 3 and 4 virus (Meng, 2010).

The geographic distribution of prevalence of HEV infection and genotypes is presented

in Figure 2.



http://www.sciencedirect.com/science/article/pii/S1201971212013197#fig0010

5

Figure 2: Geographical distribution of prevalence of human hepatitis E and genotypes. A) worldwide

prevalence of HEV infections; B) worldwide distribution of HEV genotypes. The colors used for each

country represent the predominant HEV genotypes of human and animal strains (mostly pigs) (Ruggeri et

al., 2013).

1.1.2 HEV Infection symptoms

HEV infection is silent in most individuals and when symptomatic, usually produces a

self-limiting icteric illness. Young adults, 15 to 30 years of age, are the most affected,

and the overall death rate is 0.5 to 3.0% (Smith et al., 2001). Progression to severe

fulminant liver failure may occur in certain high-risk groups, such as pregnant women

and elderly patients with underlying liver disease (Sridhar et al., 2015).

The clinical outcomes associated with HEV infection are quite diverse. The infection

most commonly manifests as self-limiting, acute icteric hepatitis, which is

indistinguishable from acute hepatitis caused by other hepatotropic viruses. In most

cases, contact with HEV leads to an asymptomatic infection followed by spontaneous

clearance of the virus, and only a minority of patients develop the symptomatic, icteric

course of the disease (Zhu et al., 2010; Rein et al., 2012).

6

Symptomatic patients experience a range of symptoms, including anorexia, jaundice,

darkened urine coloration, hepatomegaly, myalgia, elevated alanine aminotransferase

(ALT) levels in the blood, and occasionally abdominal pain, nausea, vomiting and fever

(Purcell & Emerson, 2008). Acute HEV infection in humans begins with a typical

incubation period of 2 weeks to 2 months, a transient viremia period with viral shedding

in the feces, a symptomatic phase lasting days to weeks, and jaundice apparent 2 to 3

weeks into the course of infection (Purcell, 2001). The severity of HEV infection is

considered dose dependent with alcohol use or other concurrent hepatic diseases as

contributing factors (Purcell, 2001).

Pregnant women especially from the Indian subcontinent and Africa are at increased

risk of contracting acute HEV infection (HEV 1 and 2) as well as developing severe

complications including acute liver failure (Shalimar & Acharya, 2013). HEV infection

in pregnant women may cause particularly severe illness, with a mortality rate of 15 to

25% (Aggarwal & Krawczynski, 2000; Smith et al., 2001). Death of the mother and

fetus, abortion, premature delivery, or death of a live-born baby soon after birth, are

common complications of hepatitis E infection during pregnancy (Smith et al., 2001).

The high pregnancy-associated mortality in HEV1 has not been reported with HEV3 or

HEV4 (Kamar et al., 2012).

There are numerous reports of persistent and chronic HEV infection in

immunocompromised patients, such as organ transplant recipients (Kamar et al., 2012)

and HIV infection (Dalton et al., 2009). Chronic HEV infection in immunosuppressed

persons has been shown to lead to chronic hepatitis and progressive liver fibrosis,

culminating in liver cirrhosis (Gerolami et al., 2008; Haagsma et al., 2008). All patients

with chronic HEV infection reported till date have been related to genotype 3 virus; no

cases of chronic hepatitis E caused by infection with genotypes prevalent in high-

endemicity countries, namely genotype 1 and 2, have been described (Aggarwal, 2011).

1.1.3 Hepatitis E virus hosts and reservoirs

A number of animals are known to serve as the natural hosts and reservoirs for HEV,

genotypes 3 and 4. First detections of HEV 3 in animals were reported in swine (Meng

et al., 1997), chicken (Haqshenas et al., 2001) and deer (Tei et al., 2003). HEV has been

genetically identified in rats (Johne et al., 2010), wild boar (Tian-Cheng et al., 2005),

monkeys (Yamamoto et al., 2012), mongoose (Nidaira et al. 2012), rabbits (Han et al.,




7

2014), ferrets (Li et al., 2015), cutthroat trout (Batts et al., 2011) and bats (Drexler et

al., 2012). All of these animal species are potentially natural hosts of HEV but just a

few of these species have been identified as true reservoirs of HEV. Defining a true

reservoir as a population in which the pathogen can be permanently maintained and

from which infection is transmitted to the defined target population (Haydon et al.,

2002), presumably only domestic swine, wild boar and deer should be regarded as true

reservoirs of zoonotic HEVs.

The non-travel-related HEV infections in industrialized countries are of zoonotic origin.

The food-borne zoonotic transmission of HEV is supported by studies in which the

relation between consumption of meat or organs from pigs (Yazaki et al., 2003) wild

boars (Matsuda et al., 2003) or deer (Tei et al., 2003) and hepatitis E.

Sequences of the swine HEV genotype 3 and 4 strains closely related to human strains

have been detected in many countries worldwide (van der Poel et al., 2001; Legrand-

Abravanel et al., 2009), and most frequently involved in countries formerly designated

as nonendemic for HEV, supporting that pigs are the reservoir of the indigenous

infections in these countries (Bouwknegt et al., 2007; Hakze-van der Honing et al.,

2011; Said et al., 2014).

HEV RNA has also been detected in wild boar in several countries (Tian-Cheng et al.,

2005; de Deus et al., 2008; Martelli et al., 2008; Mesquita et al., 2014), and in deer (Tei

et al., 2003; Reuter et al., 2009). More direct evidence of zoonotic food-borne

transmission of genotype 3 was obtained when four cases of hepatitis E were linked

directly to eating raw deer meat since identical HEV strains were found in the deer meat

consumed and the patients (Tei et al., 2003).

1.2 Environmental routes of HEV Transmission

The presence of human and animal pathogenic enteric viruses in water environments

reflects fecal contamination and indicates a risk to public health. Water is an important

vehicle for the transmission of enteric viruses. Rivers, lakes, streams and coastal waters

are regularly contaminated by septic tanks, storm water, overflow and agricultural and

animal manure runoff, used in agriculture, and effluents from inefficiently operated

sewage treatment plants (Lazic et al., 2015). Additionally, water could be also

8

contaminated from overflows of treatment plants impacted by flooding events, or

through direct inflow of untreated sewage (Lazic et al., 2015).

The resulting viral contamination of sea and coastal water, rivers and other surface

waters, ground waters, and irrigated vegetables and fruits is associated with subsequent

risks of reintroduction of the viral pathogens into human and animal populations (Yates

et al., 1985; Metcalf et al., 1995; Koopmans et al., 2002).

1.2.1 Sewage and Animal Manure Runoff

In areas where swine are raised, swine manure could be a source of HEV contamination

of irrigation water or coastal waters with concomitant contamination of produce or

shellfish (Smith et al., 2001). Infectious HEV has been found in swine manure and

wastewater (McCreary et al., 2008), therefore, application of manure to land and

subsequent runoff could contaminate surface water (Pina et al., 1998; Rutjes et al.,

2009).

1.2.2 Surface Water

Surface waters may also expose humans to HEV (Rutjes et al., 2009). Surface water is

easily contaminated by stable fecal-shed viruses such as HEV and acts as a public health

hazard (Rodriguez-Lazaro et al., 2012). The quality of surface water directly affects

populations utilizing the source since drinking water, and intensive farming practices

lead to higher detection rates of viruses within these sources (Rodriguez-Lazaro et al.,

2012).

Contaminated surface waters may enter food production chains, in particular via

shellfish culture areas and irrigation waters. HEV contamination of irrigation and

drinking water via animal manure or sewage, with concomitant contamination of

vegetables, fruits, or shellfish, may implicate a food safety risk (Le Guyader et al.,

2009).

Autochthonous HEV cases in no endemic regions may be associated to contaminated

fresh products. More recently HEV sequences have been detected on soft fruits and

vegetables, with irrigation water as the suspected contamination origin (Brassard et al.,

2012; Maunula et al., 2013).

9

The occurrence of a swine-like HEV genotype 3 in freshwater has also been reported in

Japan and South Korea (Li et al., 2007; Kamar et al., 2012). In Italy, HEV was detected

in surface water analyzed, with all isolates belonging to genotype 3 (Idolo et al., 2013).

Recently HEV has been detected in river waters from Italy where the sequences were

similar to sequences detected previously in patients with autochthonous HEV (no travel

history) and in animals (swine) (Iaconelli et al., 2015)

1.2.3 Coastal Water

Bivalve shellfish are at constant risk of being exposed to enteric virus as a consequence

of contamination of the shellfish beds with human or animal waste originating from

sewage treatment plants or slurry fertilized fields (Krog et al., 2014). Coastal waters

may also be contaminated by HEV leading to accumulation of the virus in the digestive

tissues of shellfish, which poses a risk of human infection through ingestion (Yugo &

Meng, 2013).

Habitually, mussels, cockles, and oysters are eaten raw or slightly cooked, thus

behaving as potential vehicles for pathogenic agents and therefore a significant health

risk. HEV is stable in both alkaline and acidic environments, after frozen for more than

10 years, and remains infectious at up to 60 °C for 1h, suggesting that a raw, rare-

cooked, or slightly steamed contaminated seafood may transmit HEV to consumers

(Emerson et al., 2005; Namsai et al., 2011;.

Shellfish consumption have been associated in an outbreak of HEV occurred onboard a

cruise ship in European waters (Said et al., 2009) and has been identified in commercial

mussels obtained from three European countries (Finland, Greece, and Spain) (Diez-

Valcarce et al., 2012) and Asia (Gao et al., 2015). Another study from Scotland showed

that from shellfish samples (mussels) analyzed 92% were tested positive for HEV RNA

genotype 3 (Crossan et al., 2012). Recently 13 different sub-genotype 4 HEV were

found in contaminated shellfish in the Bohai Gulf rim, in China (Gao et al., 2015).

There is an increased risk for travelling to regions of endemicity of acquiring HEV

infection from contaminated water and seafood, but industrialized countries are not

excepted (Zuckerman, 2003). Case reports of hepatitis E in England, Italy, and France

reveal shellfish consumption as a common source risk factor of infection (Cacopardo et

al., 1997; Ijaz et al., 2005; Renou et al., 2008).

10

1.3 Foodborne transmission

The foods related to virus contamination can be divided in two different groups

concerned to its route of contamination. One group comprises bivalve shellfish such as

oysters, clams and mussels which are contaminated with enteric viruses in their growing

sea life ( Li et al., 2007, Donia et al., 2012, Suffredini et al., 2014, Gao et al., 2015) and

the other group includes other types of foods, which are secondarily contaminated with

enteric viruses, mainly from infected food handlers, during food processing and/or food

serving (Rodríguez-Lazaro et al., 2012). In general, food may be contaminated at

different stages of production, such as by fecal contamination of shellfish-growing

waters, the use of night soil to fertilize crops, the fecal contamination of water used to

wash fruits after harvest (Le Guyader et al., 2008).

Zoonotic source for HEV infections, exposure to reservoirs of HEV might occur

through contact with animal products. The consumption of contaminated food or

drinking water (genotypes 1 and 2), may also expose humans to HEV (Rutjes et al.,

2009; Colson et al., 2010; Purcell & Emerson, 2010).

Foodborne transmission of HEV was first demonstrated in clusters of Japanese patients

after eating raw or undercooked meat from swine, wild boar or Sika deer ( Yazaki et al.,

2003; Takahashi & Okamoto, 2014;) . The genomic sequences of HEV identified from

the infected patients were identical to those recovered from the frozen leftover meat

(Tei et al., 2003; Takahashi & Okamoto, 2014).

Consumption of undercooked or raw organs or tissues such pork liver or wild boar and

sausages from infected swine has been epidemiologically linked to numerous cases of

hepatitis E worldwide (Tian-Cheng et al., 2005; Masuda et al., 2005; Meng,

2011;Miyashita et al., 2012). For example, three cases of hepatitis E in Japan were

associated with the consumption of undercooked or raw pork presumably from the same

barbeque restaurant (Miyashita et al., 2012), evidencing zoonotic foodborne HEV

transmission.

1.4 Presence of HEV in shellfish

Bivalve mollusks, such as mussels and oysters, are one of the most common foods

implicated in the transmission and dissemination of a wide variety of human enteric

viruses around the world (Lees, 2000; Polo et al., 2010). In one study, Lees (2000)

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11

observed that shellfish grown in sewage polluted waters tend to bioaccumulate

environmentally stable enteric viruses, such as norovirus, hepatitis A virus and

enterovirus. Shellfish contamination can be attributed to its contact to human fecal

pollution in the waters or virus contamination at any point during cultivation,

harvesting, processing, distribution, sale or service (Lees 2000). Bivalve mollusks filter

large volumes of water undertaken as part of their feeding process, and must

concentrate viral particles during this process, principally in the pancreatic tissue, also

called digestive diverticula, (Le Guyader et al., 2009) posing a risk of human infection

through ingestion (Yugo & Meng, 2013).

The fact of mussels, cockles, and oysters be consumed habitually raw or slightly cooked

and whole, including digestive tissues (where viruses are mainly concentrated), the

traditional way of consuming shellfish, represents a very important vehicle from

gastrointestinal infections (Donia et al., 2012) making the bivalve mollusks a high-risk

food group (Romalde et al., 1994).

It was generally thought that shellfish act as mere filters or ionic traps, passively

concentrating particles such as bacteria or virus (Le Guyader et al., 2009). However,

unlike enteric bacterial species, enteric viruses persist in shellfish for an extended period

of time and it is this persistence that appears to result in its significant impact on public

health (Le Guyader et al., 2009). HEV is deemed to be inactivated during processing

procedures used to prepare mussels for consumption, however, HEV is only 50%

inactivated at 56°C and 80% at 60°C for 1 hour, and some strains seems to be more

stable than others (Emerson et al., 2005). It is stable when exposed to

trifluorotrichloroethane, and it is resistant to inactivation by acidic and alkaline

conditions (Emerson et al., 2005).

HEV has been reported in shellfish collected in some European and Asian countries (Li

et al., 2007; Crossan et al., 2012; Donia et al., 2012; Gao et al., 2015) and years ago an

outbreak that occurred in a cruise ship in 2008 (Said et al, 2009) was related to

consumption of shellfish. One of the most difficulties to detect HEV, as well other

virus, in shellfish, is the fact that these viruses are present in very low numbers.

Nevertheless, they are present in sufficient quantities to pose a health risk (Le Guyader

et al., 2009).

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For the detection of enteric viruses in shellfish, molecular methods such as reverse

transcription-polymerase chain reaction (RT-PCR) are widely used (Le Guyader et al.

2000; Bosch et al., 2011) providing the tools for monitoring control quality and safety

of enteric viruses in shellfish as well as surveillance of outbreaks of gastroenteritis and

infectious hepatitis (Le Guyader et al. 2000; Lees 2000).

For bivalve mollusks, dissected digestive diverticulum (digestive gland) is used as the

starting material with further enzymatic digestion using proteinase K combined with

heat treatment at 65 °C (Jothikumar et al., 2005). This enzymatic/thermal treatment

damages the viral capsid, thereby releasing the nucleic acids (Nuanualsuwan & Cliver,

2002). Focusing the analysis of shellfish on the digestive tissues enhances assay

performance by eliminating tissues (i.e. adductor muscle) that are rich in inhibitors

(Atmar et al. 1995). However, shellfish tissue identification using PCR technique still

presented with several problems. The low quantity of virus in shellfish samples render

them a difficult and variable matrix that is also known to cause amplification inhibition

(Lowther et al., 2008) increasing the risk of false negative results (Diez-Valcarce et al.,

2012). For this reason, effective preliminary sample treatment steps such as elution and

concentration of viruses from the shellfish tissue and RNA extraction and purification

are essential for final PCR accuracy and reproducibility (Le Guyader et al., 2000).

1.5 Detection of HEV in food

Polymerase chain reaction-PCR-based methods have been successfully used to monitor

food products for viral contamination (Rodriguez et al., 2009). During PCR, a fragment

of the viral genome is amplified using specific primers. For RNA viruses, RT of the

viral RNA to a cDNA strand is necessary prior to the PCR.

1.5.1 Real-time PCR

The development of real-time PCR has represented one of the most significant advances

in food diagnostics as it provides rapid, reliable and quantitative results. These features

become increasingly important for the agricultural and food industry. Different

strategies for real-time PCR diagnostics have been developed including unspecific

detection independent of the target sequence using fluorescent dyes such as Sybr Green,

or by sequence-specific fluorescent oligonucleotide probes such as TaqMan® probes


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(Rodriguez-Lazaro & Hernandez, 2013). In both cases, fluorescence is measured during

each cycle, and when the amount of fluorescence exceeds the background level

(threshold level), the sample is scored as positive (Rodriguez et al., 2009). The number

of cycles needed to reach the threshold level, commonly referred to as the cycle

threshold value, correlates with the amount of target in the sample prior to amplification

(Rodriguez et al., 2009).

Some enteric viruses, like HEV, do not grow in any cell culture-based detection system

(Duizer et al. 2004) consequently a real-time PCR was raised as an elective method for

the detection of the presence of these viruses in food, water or environmental samples.

The analysis of the presence of virus in food is an integral part of food quality control,

as well as of the management of food chain safety (De Medici et al., 2015).

Real-time quantitative PCR (qPCR) is very similar to traditional PCR. In qPCR the

amount of DNA is measured after each cycle via fluorescent dyes that yield increasing

fluorescent signal in direct proportion to the number of PCR product molecules

(amplicons) generated (Sigma Aldrich, 2008). Data is collected in the exponential phase

of the reaction yield. The major difference being that with qPCR the amount of PCR

product is measured after each round of amplification while with traditional PCR, the

amount of PCR product is measured only at the end point of amplification (Sigma

Aldrich, 2008).

HEV is a single stranded RNA virus and therefore require reverse transcription to be

converted into double-stranded cDNA prior to PCR. When this stage is necessary, the

capacity of an extraction method to obtain a nucleic acid sample as pure as possible is a

particularly important point. Indeed, the high susceptibility of reverse transcriptase to

inhibitory substances is a major limiting factor in such methods (De Medici et al.,

2015).

The reverse transcriptase is as critical to the success of qRT-PCR as the DNA

polymerase once it introduces substantial variation into a qRT-PCR assay (Bustin et al.,

2009). It is important to choose a reverse transcriptase that not only provides high yields

of full-length cDNA, but also has good activity at high temperatures. High-temperature

performance is also very important for denaturation of RNA with secondary structure

(Life technologies, 2012).

14

Many studies have been used a real time RT-PCR developed in 2006, designed by

Jothikumar et al., (2006) to detect the 4 main genotypes of HEV in different types of

matrices (Crossan et al., 2012; Son et al., 2014). This broadly reactive TaqMan®

RT-

PCR assay that use primers that target a conserved region of the ORF 3, was proposed

for HEV detection in clinical and environmental samples but that have been also used

for food matrices (Bartolo et al., 2015).

1.5.2 Real-time PCR design

When a qPCR assay is designed, the most important parameters are the amplicon length

and the melting temperature (Tm) of the primers and probe. The optimal amplicon

length should be less than 150 bp, but it is advisable to reduce the length below 80 bp.

However, amplicons up to 300 bp amplify efficiently (Rodriguez-Lazaro & Hernandez,

2013). Shorter amplicons amplify more efficiently than longer ones and are more

tolerant to suboptimal reaction conditions. This is because they are more likely to be

denatured during the 92-95°C PCR step, allowing the probes and primers to compete

more effectively for binding to their complementary targets. As the extension rate of

Taq polymerase is between 30 and 70 bases per second (Jeffreys et al., 1988),

polymerization times as short as 5 s are sufficient to replicate such amplicon, making

amplification of artefacts less likely and reducing the time of the assay (Rodriguez-

Lazaro & Hernandez, 2013).

Primers

Good primer design is one of the most important parameters in real-time PCR. Primers

should be designed according to standard PCR guidelines. They should be specific for

the target sequence and be free of internal secondary structure (Life technologies, 2012).

Primer pairs should have compatible melting temperatures (within 5°C) and contain

approximately 50% guanine (G) and cytosine (C) content. Primers with high GC

content can form stable imperfect hybrids. Conversely, high AT (adenine and thiamine)

content depresses the Tm of perfectly matched hybrids. If possible, the 3’ end of the

primer should be GC rich (GC clamp) to enhance annealing of the end that will be

extended (Life technologies, 2012).

15

The primer pair sequences must be analyzed to avoid complementarity and

hybridization between primers (primer-dimers) (Life technologies, 2012).

Primers are generally used in the 50–300 nM range. Higher concentrations may promote

mispriming and accumulation of non-specific products, and lower concentrations may

lead to primer exhaustion, although target copy numbers will have been calculated well

before. Non-specific priming can be minimized by selecting primers that have only one

or two G/Cs within the 3′ last five nucleotides (Rodriguez-Lazaro and Hernandez,

2013). A relative instability at the 3′ ends makes primers less likely to hybridize

transiently causing non-specific extension (Rodriguez-Lazaro & Hernandez, 2013).

Probes

There are different types of specific-sequence fluorescent probes, and they can be

classified into two major groups, hydrolysis probes and hybridization probes, both types

being homologous to the internal region amplified by the two primers. The fluorescence

signal intensity can be related to the amount of PCR product (i) by a product-dependent

decrease of the quench of a reporter fluorophore or (ii) by an increase of the FRET

(Fluorescence Resonance Energy Transfer) from a donor to an acceptor fluorophore.

The FRET and the quench efficiency are strongly dependent on the distance between

the fluorophores. Therefore, the PCR-product-dependent change in the distance between

the fluorophores is used to generate the sequence-specific signals. Several different

formats can be used. In principle, all of them could function by a decrease of quench or

an increase of FRET; in practice, most formats are based on a decrease of quench

(Rodriguez-Lazaro & Hernandez, 2013).

The most commonly used fluorescent reporter dyes are FAM (6-car-boxyfluorescein),

TET (tetrachloro-6-car-boxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxy-

fluorescein) or HEX (hexacholoro-6-carboxyfluorescein), and the most frequently used

quenchers are TAMRA (5-Carboxytetramethylrhodamine), DABCYL (4-((4-

(dimethylamino) phenyl)azo) benzoic acid) and Black Hole Quencher (BHQ). Many

commercial fluorophores have been developed by biotech companies such as VIC,

Alexa Fluor, or Yakina Yelow (Rodriguez-Lazaro & Hernandez, 2013).

The hydrolysis probes are cleaved when hybridized by the 5′-3 exonuclease activity of

particular DNA polymerases (Holland et al., 1991) during the elongation phase of

16

primers, yielding a real time measurable fluorescence emission directly proportional to

the concentration of the target sequence (Heid et al., 1996). The best known hydrolysis

probes are TaqMan® probes and TaqMan

® MGB (minor groove binder) probes, both

developed by Applied Biosystems (Rodriguez-Lazaro & Hernandez, 2013).

A TaqMan® probe is an oligonucleotide double-labelled with a reporter fluorophore at

the 5′ end (reporter dye) and with a quencher internally or at the 3′ end (quencher dye).

In addition, the probes must be blocked at their 3′- end to prevent the extension during

the annealing step. The TaqMan®

assay (Figure 3) uses three oligonucleotides. Two

conventional primers allow amplification of the product, to which the TaqMan® probe

will anneal (Rodriguez-Lazaro & Hernandez, 2013). The quencher dye absorbs the

fluorescence of the reporter dye due to its proximity, which permits FRET. When the

correct amplicon is amplified, the probe can hybridize to the target after the

denaturation step. It remains hybridized while the polymerase extends the primers until

it reaches the probe. Then, it displaces its 5′ end to hold it in a forked structure. The

enzyme continues to move from the now free end to the bifurcation of the duplex, where

cleavage takes place (Lyamichev et al. 1993). The quencher is hence released from the

fluorophore, which now fluoresces after excitation (Heid et al., 1996; Holland et al.,

1991; Gibson et al., 1996). As the polymerase will cleave the probe only while it

remains hybridized to its complementary strand, the temperature conditions of the

polymerization phase of the PCR must be adjusted to ensure probe binding. Most

probes have a Tm of around 70°C; therefore, the TaqMan® system uses a combined

annealing and polymerization step at 60-63°C. This ensures that the probe remains

bound to its target during the primer extension step. It also ensures maximum 5′-3′

exonuclease activity of the Taq and Tth DNA polymerases (Tombline et al. 1996).

The TaqMan® MGB probes are similar to TaqMan

® probes. They contain a non-

fluorescent quencher and an oligopeptide at the 3′ end. This oligopeptide is a DNA

MGB, with very high affinity for the minor groove of AT-rich double-stranded DNA

(Afonina et al., 1997). Addition of the MGB ligand significantly enhances duplex

stability. The shorter the probe, the greater the MGB contribution to the overall duplex

stability (Rodriguez-Lazaro & Hernandez, 2013).

17

Figure 3: Real-time PCR detection using TaqMan® probes 5’-3’ polymerase and exonuclease activity of

the Taq DNA polymerase (TAQ). R: reporter; Q: quencher (Rodriguez-Lazaro & Hernandez, 2013).

In contrast to hydrolysis probes, hybridization probes are not hydrolysed during PCR.

The fluorescence is generated by a change in its secondary structure during the

hybridization phase, which results in an increase of the distance separating the reporter

and the quencher dyes. The most relevant hybridization probes are those containing

hairpins (Molecular Beacons, Scorpion primers, etc.), and FRET hybridization probes


The Ta (annealing temperature) of the probe is also a critical parameter since

amplification primers are extended as soon as they bind to their targets. The

hybridization target sequence is rapidly masked with newly synthesized DNA.

Therefore, the Ta of the probes must be significantly greater (approximately 10°C) than

that of the primers. The presence of G at the 5′ end of the probe is to be avoided,

because it slightly quenches the reporter signal, even after probe cleavage. Furthermore,

the probe should contain more Cs than Gs; if this is not the case, the antisense probe

should be used. The probe should never overlap with, or be complementary to either of

the primers. The optimum concentration of fluorogenic probes will vary with the type of

probe, as it depends on background fluorescence: quenching of hydrolysis probes is

often below 100%, and thus they produce background fluorescence levels higher than

molecular beacons and FRET probes (Rodriguez-Lazaro & Hernandez, 2013).


18

Sybrgreen

Dye-based detection is performed via incorporation of a DNA binding dye in the PCR.

The dyes are non-specific and bind to any double-stranded DNA (dsDNA) generated

during amplification resulting in the emission of enhanced fluorescence. This allows the

initial DNA concentration to be determined with reference to a standard sample (Sigma

Aldrich, 2008).

DNA binding dyes bind reversibly, but tightly, to DNA by intercalation. Most real-time

PCR assays that use DNA binding dyes detect the binding of the fluorescent binding

dye SYBR® Green I, or the more stable binding dye SYBR

® Gold, to DNA (Sigma

Aldrich, 2008).

Prior to binding DNA, these dyes exhibit low fluorescence. During amplification,

increasing amounts of dye bind to the double stranded DNA products as they are

generated. For SYBR Green I, after excitation at 497 nm (SYBR Gold 495 nm), an

increase in emission fluorescence at 520 nm (SYBR Gold 537 nm) results during the

polymerization step followed by a decrease as DNA is denatured. Fluorescence

measurements are taken at the end of the elongation step of each PCR cycle to allow

measurement of DNA in each cycle. The Figure 4 illustrates how a dye based assay

works. Assays using SYBR Green I binding dye are less specific than conventional

PCR with gel detection because the specificity of the reaction is determined entirely by

the primers. However, additional specificity can be achieved and the PCR can be

verified by melt or dissociation curves (Sigma Aldrich, 2008).

19

Figure 4: Real-time PCR detection using Sybr Green® (Rodriguez-Lazaro & Hernandez, 2013).

Melt curves allow a comparison of the melting temperatures of amplification products.

Different dsDNA molecules melt at different temperatures, dependent upon a number of

factors including GC content, amplicon length, secondary and tertiary structure, and the

chemical formulation of the reaction chemistry. To produce melt curves, the final PCR

product is exposed to temperature gradient from about 50°C to 95°C while fluorescence

readouts are continually collected. This causes denaturation of all dsDNA. The point at

which the dsDNA melts into ssDNA is observed as a drop in fluorescence as the dye

dissociates. The melt curves are converted to distinct melting peaks by plotting the first

negative derivative of the fluorescence as a function of temperature (-dF/dT). Products

of different lengths and sequences will melt at different temperatures and are observed

as distinct peaks (Sigma Aldrich, 2008).

1.5.3 Analytical verification

Standard curve

A dilution series of known template concentrations can be used to establish a standard

curve for determining the initial starting amount of the target template in experimental

samples or for assessing the reaction efficiency. The log of each known concentration in

the dilution series (x-axis) is plotted against the Ct value for that concentration (y-axis).

From this standard curve, information about the performance of the reaction as well as

20

various reaction parameters (including slope, y-intercept, and correlation coefficient)

can be derived. The concentrations chosen for the standard curve should encompass the

expected concentration range of the target in the experimental samples (Life

technologies, 2012).

Efficiency

PCR amplification efficiency must be established by means of calibration curves,

because such calibration provides a simple, rapid, and reproducible indication of the

mean PCR efficiency, the analytical sensitivity, and the robustness of the assay (Bustin

et al., 2009).

A PCR efficiency of 100% corresponds to a slope of -3.32, as determined by the

following equation: Efficiency = 10(-1/slope)

-1. Ideally, the efficiency (E) of a PCR

reaction should be 100%, meaning the template doubles after each thermal cycle during

exponential amplification. The actual efficiency can give valuable information about the

reaction. Experimental factors such as the length, secondary structure, and GC content

of the amplicon can influence efficiency. Other conditions that may influence efficiency

are the dynamics of the reaction itself, the use of non-optimal reagent concentrations,

and enzyme quality, which can result in efficiencies below 90%. The presence of PCR

inhibitors in one or more of the reagents can produce efficiencies of greater than 110%.

A good reaction should have an efficiency between 90% and 110%, which corresponds

to a slope of between -3.58 and -3.10 (Life technologies, 2012).

Correlation coefficient (R2)

The R2 value of a standard curve represents how well the experimental data fit the

regression line, that is, how linear the data are. Linearity, in turn, gives a measure of the

variability across assay replicates and whether the amplification efficiency is the same

for different starting template copy numbers. A significant difference in observed Ct

values between replicates will lower the R2 value (BioRad, 2006).

Normalization (validation)

The primer-dimer formation should be checked by analyzing a well-documented sample

(reference material, e.g. proficiency testing sample) with melt-curve analysis, resulting

in one single peak. The size of the amplicon, analyzed by gel electrophoresis, should be

21

of the expected size. The product amplified must be analyzed by sequence analysis,

followed by a comparison of the target sequence with sequences in Genbank

(Raymaekers et al., 2009).

1.6 Internal Quality Control: Controls and inhibitors of PCR assays

Although the detection of enteric viruses in food is mainly done by molecular

techniques, there are several limitations. Polymerase chain reaction is considered a very

sensitive technique and is well recognized to be susceptible to cross-contamination

events within the laboratory and also to matrix interferences causing PCR inhibition

(Bustin et al., 2009) favoring false negative results and demonstrating the need for

proper quality control (Bosch et al., 2011).

In general, quality control of methods for detection of viruses in food samples implies

the use of adequate controls throughout the different steps that are considered critical

for correct detection (Stals et al., 2012). For molecular detection, the use of negative

and positive controls has been reviewed elsewhere.

During virus detection, false negative results, false positives, or both can occur. False

negative results are caused by inhibition and false positives can occur because of cross-

over contamination. The risk for cross-over contamination rises when using a highly

sensitive molecular method (Rijpens & Herman, 2002). For this reason, appropriate

positive and negative controls should be included (Stals et al, 2012).

Positive controls in the form of nucleic acids extracted from experimental samples are

useful for monitoring assay variation over time and are essential when calibration

curves are not performed in each run (Bustin et al., 2009).

A negative control is an aliquot of highly pure water used as template in a real-time RT-

PCR reaction to control for contamination in the real-time RT-PCR reagents (Bosch et

al., 2011). A negative control should be included in each PCR-based method for food

control. This is prepared in a separate tube and contains all PCR components with the

exception of any DNA template. The results of this reaction should be always negative.

An accidental positive result would indicate contamination of working solutions, tubes

or pipette tips by DNA (De Medici et al., 2015).

22

1.6.1 Process Controls

For monitoring of food supply chain for viruses is necessary that the analytical results

can be reliably verified. It is essential therefore that verification includes recognition of

analyses where the method has failed to perform correctly, as this may mask the

presence of a virus in a sample by a false-negative interpretation of the absence of a

signal (Diez-Valcarce et al., 2011). Incorrect performance can occur during the sample

treatment or the assay, and failed methods can be identified by the use of a sample

process control (SPC) (Diez-Valcarce et al., 2011).

A process control consists of adding a control virus to a (parallel) tested sample (Stals et

al., 2012). The addition of this control will verify that pre-amplification sample

treatment has worked correctly, and identify those samples in which pre-amplification

sample treatment has failed as well as enable the determination of the method's

efficiency of detection (Diez-Valcarce et al., 2011).

A process control should be used to indicate the effect of the food matrix on the virus

extraction efficiency (Stals et al., 2012). This may be very useful due to the great

variety of foods at risk for viral contamination (Stals et al., 2012). The use of an

appropriate process control has been also debated for the evaluation of the rate of

recovery in the quantitative detection of viruses in different food matrices (De Medici et

al., 2015). The murine norovirus 1 (MNV-1), the feline calicivirus, a genetically

modified mengovirus, and the MS2 bacteriophage have most frequently been used as

process control for detection of enteric viruses in food samples (Stals et al., 2012).

1.7 Inhibition of PCR assays

Although the detection of enteric viruses in food is mainly done by molecular

techniques, there are several limitations. The method is susceptible to inhibition,

favoring false negative results and demonstrating the need for proper quality control.

Many matrices from the food supply chains most susceptible to virus contamination like

soft fruit, salad vegetable and shellfish, are complex and difficult to treat, and can

furthermore contain substances which can inhibit nucleic acid amplification or reduce

amplification efficiency (Diez-Valcarce et al., 2011). The PCR inhibitors may act

through one or more of the following mechanisms: interference with the cell lysis step,

23

degradation or capture of the nucleic acids, or inactivation of the thermostable DNA

polymerase (Wilson, 1997).

Common inhibitors include various components food constituents (e.g., organic and

phenolic compounds, proteinase, glycogen, fats, and Ca2+

) (Tsai & Olson, 1992), and

environmental compounds (e.g., phenolic compounds, humic acids, and heavy metals)

in shellfish, acidic polysaccharides and glycogen (Lee et al., 1995; Wilson, 1997;

Blackstone et al., 2003; Kaufman et al., 2004).

For enteric RNA viruses, a reverse transcription stage is necessary, the capacity of an

extraction method to obtain a nucleic acid sample as pure as possible is a particularly

important point. Indeed, the high susceptibility of reverse transcriptase to inhibitory

substances is a major limiting factor in such methods (De Medici et al., 2015).

Several ways have been described to overcome this inhibition, such as the analysis of

samples dilutions, smaller sample sizes, adaptation of the PCR by, e.g., the addition of

Tween, BSA (Bovine Serum Albumin), or commercial reagents (Bosch et al., 2011)

providing a simple method that can facilitate amplification, albeit with reduced

sensitivity (Wilson, 1997).

The sample preparation and DNA/RNA extraction steps should aim at minimizing

inhomogeneity through achievement of a certain level of robustness and analyte quality

that should be mainly characterized by analyte purity and integrity (De Medici et al.,

2015).

24

2. OBJECTIVES

____________________________________________________________

25

2. OBJECTIVES

The aims of the present work were:

1. To search for the presence of HEV RNA in shellfish samples.

For this purpose, a total of 81 shellfish samples collected in coastal water of Peninsula

Iberian, Spain, were tested for the presence of HEV RNA using three in house real-time

RT-PCR approaches (two using probe and one using Sybr® Green) targeting a

conserved region within the ORF 3 of the HEV genome.

2. To characterize the HEV positive samples in order to evaluate their

genetic relationship with strains already known.

For that purpose the shellfish samples that showed to be positive for HEV RNA by real-

time RT-PCR (using probe and/or Sybr® Green) were tested by nested RT-PCR

targeting the ORF1 region within the HEV genome.

26

3. MATERIALS AND METHODS

______________________________________________________________________

27

3. Material and Methods

3.1 Samples

In total 81 mussels (Mytilus galloprovincialis) batch processed (by the team of

Professor Jésus Romalde from the Microbiology and Parasitology Department,

University of Santiago Compostela, Spain), according to the developed standard method

ISO/TS 15216-1:2003 were studied. All the samples were spiked with mengovirus

before nucleic acid extraction, in order to evaluate the extraction efficiency.

It was the nucleic acid extracted from these samples that were used in the present study.

3.1.1 HEV and mengovirus

A stock solution of HEV (108 RNA copies/µl) was used to generate the standard curves

necessary to calculate the detection limit of real-time RT-PCR assay. It was also used

as a positive control in real-time RT-PCR reactions.

A stock solution of mengovirus (109

RNA copies/µl) was used to calculate the

extraction efficiency.

These stock viral solutions were kindly provided by Professor Romalde.

3.2 Real-time RT-PCR for detection of HEV RNA

Three in house real-time RT-PCR approaches were performed. Two using probe

(Qiagen OneStep RT-PCR and Kapa Fast Universal One-Step qRT-PCR) and one using

Syber Green (One Step Kapa Sybr®

Fast qPCR).

3.2.1 Primers and probe

The primers and probe used in real-time RT-PCR, target a conserved region in ORF3,

allowing the detection of different genotypes of HEV (Jothikumar et al., 2006). The

sequences of forward primer (JVHEVF) reverse primer (JVHEVR) and probe

(JVHEVP) are showed in Table 1. The TaqMan® probe contains a 5’ 6-carboxy

fluorescein fluorophore and 3’ black hole quencher. The sequences position corresponds

to 5261-5330 nt of the HEV genome.

28

Table 1: Primers and probe used for the detection of HEV RNA by real-time RT-PCR.

Target

virus

Name

primer/probe

Primer/probe Sequence 5´- 3´ Sense Sequence

position

Reference

HEV

JVHEVF GGTGGTTTCTGGGGTGAC + 5261-5330

Jothikumar

et al., (2006) JVHEVR AGGGGTTGGTTGGATGAA - 5261-5330

JVHEVP TGATTCTCAGCCCTTCGC 5285-5302

3.2.2 Qiagen OneStep RT-PCR

The Qiagen OneStep RT-PCR kit (Qiagen®, Hilden, Germany) was adapted for real-

time RT-PCR assay. In this assay the reverse transcription and the PCR amplification

of the cDNA is carried out consecutively in the same tube. . From now this kit will be

designated as Qiagen qRT-PCR.

The 81 shellfish samples (nucleic acid extracts) were tested undiluted and diluted 1:10

(in RNasefree and DNase free water) with the Qiagen qRT-PCR according to the

manufacturer's protocol. Briefly, 10 μl of each extract sample was added to the

mastermix consisting of 5 µl Qiagen OneStep RT-PCR buffer, 0.4 mM dNTP mix, 1 μl

Enzyme Mix, 250 nM of each primer and 100 nM of TaqMan® probe (Table 2). The

final reaction volume was 25 μl.

Table 2: Mastermix for detection of HEV RNA by Qiagen qRT-PCR.

Final concentration Volume for 25μl (µl)

Qiagen OneStep RT-PCR buffer (5X) 1x 5,0

Primer forward 250 nM 0,625

Primer reverse 250 nM 0,625

Probe 100 nM 1,25

RNase-free water - 5,5

dNTP mix 0.4 mM 1,0

Qiagen OneStep RT-PCR Enzyme Mix - 1,0

Template - 10,0

The thermal conditions consisted of an initial RT at 50 °C for 15 min followed by a

initial PCR activation at 95ºC for 10 min, and 45 cycles of sample denaturation at 95°C

for 15 sec and primer annealing-extension at 60ºC for 1 min (Table 3). All amplification

29

steps were performed in a real-time thermo cycler, MiniOpticon System (Bio-rad

Laboratories).

To minimize potential contamination mastermix and preparation the addition of the

samples were performed in separate rooms. In all the assays a positive control (HEV

RNA stock solution) and a negative control (RNase free water) were included. The

negative control were added at the same room as mastermix.

Table 3: Thermal conditions for Qiagen qRT-PCR.

Step Time/ temperature

Reverse transcriptase 15 min/50 °C

Initial PCR activation 10 min/95ºC

Denaturation 15 sec/95 °C

Annealing/extension 60 sec/60ºC

PCR cycles 45

Detection limit of the Qiagen qRT-PCR assay

In order to evaluate the detection limit of the Qiagen qRT-PCR a standard curve was

generated using a 10 fold dilution of the HEV stock solution (concentration of 109

RNA

copies/μl), ranging from 4x109 to 4x10

0 copies/reaction.

3.2.3 Kapa Fast Universal One-Step qRT-PCR

The Kapa Fast Universal One-Step qRT-PCR kit (Kapa Byosistems®,

Massachusetts,USA) is also an one-step real-time RT-PCR. From now this kit will be

designated as Kapa qRT-PCR .

Only 21 shellfish samples (nucleic acid extracts) were tested with Kapa qRT-PCR.

Samples were tested undiluted according to the manufacturer's protocol. Briefly, 5 μl of

each RNA extract sample was added to 15 μl of mastermix containing 250 nM of

primers and 100 nM of TaqMan®

probe, 0.4 μl of enzyme RT mix (Table 4).The final

reaction volume was 20 μl.

30

Table 4: Mastermix for detection of HEV RNA by Kapa qRT-PCR.


Kapa Fast Master Mix (2X) 1x 10,0



Probe 100 nM 1,0

PCR grade water - 3,4

Kapa RT Mix (50x) 1x 0,4

Template - 5,0

The thermal conditions consisted of an initial RT at 42 °C for 5 min followed by a

initial PCR activation at 95ºC for 5 min, and 40 cycles of sample denaturation at 95°C

for 3 sec and primer annealing-extension at 60ºC for 20 sec (Table 5). All amplification

steps were performed in a real-time thermo cycler, MiniOpticon System (Bio-rad

Laboratories).





Table 5: Thermal conditions for Kapa qRT-PCR.

Step Time/Temperature

Reverse transcription 5 min/42ºC

PCR activation 5 min/95ºC


Annealing/extension 20 sec/60ºC

PCR cycles 40

Detection limit of the Kapa qRT-PCR assay

In order to evaluate the detection limit of the Kapa qRT-PCR a standard curve was

generated using a 10 fold dilution of the HEV stock solution (concentration of 108 RNA


-1 copies/reaction.

31

3.2.4 One Step Kapa Sybr® Fast qPCR

The one Step Kapa Sybr® Fast qPCR (Kapa Biosystems, Massachusetts, USA), that is

also an one-step real-time RT-PCR was performed according to the manufacturer's

protocol. From now this kit will be designated as Kapa Sybr®

qPCR.

A total of 33 shellfish samples (nucleic acid extracts) were tested undiluted and diluted

(1:5) (in RNasefree and DNase free water) with the Kapa Sybr® qPCR according to the

manufacturer's protocol. Briefly, 5 μl of each RNA extract sample was carried out in a

15 μL reaction which consisted of 5 μL viral RNA, 10 μL Kapa Sybr Fast qPCR Master

Mix (2x), 300 nM of each forward and reverse primers and 0.4 μL of Kapa RT Mix.

The final reaction volume was 20 μl (Table 6).

Table 6: Mastermix for detection of HEV RNA by Kapa Sybr® qPCR.


Kapa Sybr Fast qPCR Master Mix (2X) 1x 10,0



PCR grade water - 4,36

Kapa RT Mix (50x) 1x 0,4

Template - 5,0

The thermal conditions consisted of an initial RT at 42 °C for 5 min followed by a PCR

activation at 95ºC for 5 min, and 40 cycles of sample denaturation at 95°C for 3 sec and

a primer annealing-extension temperatures of 60ºC (condition A) or 64ºC (condition B)

for 20 sec (Table 7). These two temperatures of annealing-extension were used in order

to find the better amplification condition. Subsequently, a melting curve was recorded

by heating at 0.2 ºC/s up to 95ºC. All amplification steps were performed in a real-time

thermo cycler, CFX96 TouchTM

(Bio-rad Laboratories). The amplification and melting

curve data were collected and analyzed using the BioRad CFX managerTM

software.





Table 7: Thermal conditions tested for optimization of HEV RNA detection by Kapa

Sybr® qPCR.

32

Step Time/temperature

A

Time/temperature

B

Reverse transcriptase 5 min/42ºC 5 min/42ºC

PCR activation 5 min/95ºC 5 min/95ºC

Denaturation 3 sec/95 °C 3 sec/95 °C

Annealing/ Extension 20 sec/60ºC 20 sec/64ºC

PCR cycles 40 40

Two different annealing temperatures were used for optimization: A) 60ºC; B) 64ºC.

Detection limit of the Kapa Sybr® qPCR. assay

In order to evaluate the detection limit of the Kapa Sybr® qPCR. a standard curve was

generated using a 10 fold dilution of the HEV stock solution (concentration of 108 RNA


-1 copies/reaction.

3.3 Nested RT-PCR for the detection of HEV RNA

3.3.1 Primers

The set of primers used target a highly conserved region of ORF1. The primers used for

the first round RT-PCR were HEV-cs and HEV-cas, and for the second round PCR was

HEV-csn and HEV-casn (Table 8). The first round consisted of an amplification of 470

bp and the second round consisted of an amplification of 330 bp within the previous

DNA segment.

Table 8: Primers used for detection of HEV RNA by nested RT-PCR.

Step Name

primer

Primer Sequence 5´-3´ Sense Sequence

position

(bp)

Reference

RT-

PCR

HEV-cs TCGCGCATCACMTTYTTCCARAA +

469-472

Johne et

al., (2010)

HEV-cas GCCATGTTCCAGACDGTRTTCCA -

Nested-

PCR

HEV-csn TGTGCTCTGTTTGGCCCNTGGTTYC†G +

331-334 HEV-casn CCAGGCTCACCRGARTGYTTCTTCCA -

D=A, G or T; M=A or C; N=A, C, G or T; R=A or G; Y=C or T.

3.3.2 Nested RT-PCR

A nested RT-PCR assay was performed in 33 shellfish samples, undiluted, diluted (1:5)

and (1:10) (in RNasefree and DNase free water). The first round, RT-PCR, was

33

performed using Qiagen OneStep RT-PCR kit (Qiagen®, Hilden, Germany). Briefly, 5

μl of each sample was added to the mastermix consisting of 0.5 µM Qiagen OneStep

RT-PCR buffer, 0.4 mM dNTP mix, 1 μl Enzyme Mix, 500 nM of each forward and

reverse primers and 0.5 μl of RNase inhibitor (Table 9). The final reaction volume was

25 μl.

Table 9: Mastermix for detection of HEV RNA by nested RT-PCR - first round.

Final concentration Volume for 25μl (μl)

Qiagen OneStep RT-PCR buffer (5X) 1x 5,0

Primer foward 500nM 0,25


RNase free water - 12,0

dNTP mix 0.4 mM 1,0

Enzyme mix - 1,0

Rnase inhibitor ~20 U 0,5

Template - 5,0

The thermal conditions comprised 50ºC for 30 min and 95ºC for 15 min, followed by 40

cycles of denaturation at 94˚C for 60 sec, annealing at 52˚C for 60 sec, extension at

72˚C for 45 sec, with a final incubation at 74ºC for 5 min (Table 10).

To minimize potential contamination mastermix preparation and the adition of the

samples or amplicons were made in a separate room. To each run a negative control

(RNase free water), which was prepared at the same room as mastermix, and a positive

control (HEV RNA) were included.

Table 10: Thermal conditions for nested RT-PCR - first round.

Step Time/ Temperature




Annealing 60 sec/50ºC

Extension 60 sec/72ºC

Final Extension 10 min/72ºC

The second round was performed using Kapa Taq DNA Polymerase kit (Kapa

Byosistems®, Massachusetts, USA). Briefly, 5 μl of the amplicons obtained in the fisrt

round RT-PCR were added to the mastermix consisting of 0.5 µM 5 x KAPA Taq

34

buffer A, 0.5U of KAPA Taq DNA Polymerase, 10 mM of dNTP mix, 0.5 μM of

forward and reverse primers The final reaction volume was 25 μl (Table 11).

Table 11: Mastermix for detection of HEV RNA by nested RT-PCR - second round.

Final concentration Volume for 25μl (μl)

KAPA Taq buffer A (10 x) 1 X 2,5

KAPA dNTP mix 0.3 mM 0,5

KAPA Taq DNA Polymerase 5U/µl 0,1

Primer foward 0.5 µM 0,2

Primer reverse 0.5 µM 0,2

Rnase-free water - 16,5

Template - 5,0

The thermal conditions comprised 95 °C for 3 min, followed by 40 cycles of sample

denaturation at 95°C for 30 sec, and then by primer annealing at 50ºC for 30 sec,

extension for 30 sec at 72ºC and final extension at 72ºC for 10 min (Table 12).

All amplification steps were performed in T100TM Thermal Cycler (Bio-Rad

Laboratories).

Table 12: Thermal conditions for nested RT-PCR - second round.


Initial denaturation 3 min/95 °C

Denaturation 30 sec/95ºC

Annealing 30 sec/50 °C


Final extension 10 min/72ºC

3.4 Electrophoresis and purification of amplified products

The final amplified products, obtained in the nested RT-PCR, were separated by

electrophoresis using 1.5% agarose gel (Seakem LE Agarose, USA) with Midori Green

Advanced DNA stain (Nippon Genetics, Germany) for 45 min., 80V. A 100 bp ladder

(GRS Ladder 100bp Grisp®, Portugal) was used. The results were visualized with the

Molecular Imager Gel Doc XR+System and Image Lab Software (Bio-Rad®, USA).

35

The amplified products obtained in one step and nested RT-PCR with the expected size

(~330 bp) were recovered from agarose gel and purified with the GRS PCR and Gel

Band Purification Kit (GRisp, Porto, Portugal), according to the manufacture’s

instruction.

3.4.1 Sequencing of HEV nested RT-PCR products

The purified amplicons were sent to Stab Vida (Caparica, Portugal) to be sequenced,

along with the corresponding primers, diluted to 10pmol/μl.

Editing and multiple alignments of sequences were performed using BioEdit version 2.1

(Ibis Biosciences, California, USA), and they were compared with all HEV sequences

of different genotype (1-4) available from GenBank with the Basic Alignment Search

Tool (BLAST). Phylogenetic analysis was performed using MEGA version 4.0 (Tamura

et al., 2007).

3.5 Real-time RT-PCR for mengovirus

3.5.1 Primers and probe

Primers and probe used, target a conserved region 5’ noncoding region of viral RNA of

mengovirus. The sequences of the forward primer Mengo 110, reverse primer Mengo

210 and the Mengo 147 probe, labeled at 5’ end with 6-carboxy fluorescein fluorophore

(FAM), and modified at the 3’ end with the addition of a minor groove binder (MGB)

are showed in Table 13. Both primers and probe were provided by Professor Romalde.

Table 13: Primers and probe used for mengovirus detection.

Target virus Name

primer/probe

Primer/probe Sequence 5´- 3´ Sense Reference

Mengo 110 GCGGGTCCTGCCGAAAGT +

Pinto et al.,

(2009)

Mengovirus Mengo 209 GAAGTAACATATAGACAGACGCACA -

Mengo 147 ATCACATTACTGGCCGAAGC

36

3.5.2 Qiagen OneStep RT-PCR

The Qiagen Onestep RT-PCR kit (Qiagen®,Hilden, Germany) was used. Only 23

shellfish samples (nucleic acid extracts) were tested. Briefly, 5 μl of each extract sample

undiluted and diluted 1:10 (in RNasefree and DNase free water) was added to 20 μl of

mastermix containing 5 µl Qiagen OneStep RT-PCR buffer, 0.4 mM dNTP mix, 1μl

Enzyme Mix, 900 nM forward primer, 500nM reverse primer and 450 nM TaqMan®

probe (Table 14). The final reaction volume was 25 μl.

Table 14: Mastermix for detection of mengovirus by Qiagen qRT-PCR.


Qiagen OneStep RT-PCR buffer (5X) 1X 5,0



Probe 100nM 450 nM 1,125

RNase-free water - 8,375

dNTP mix 0.4 mM 1,0

Qiagen OneStep RT-PCR Enzyme Mix - 1,0

Template 5,0

The thermal conditions were for the reverse transciption 55˚C during 60 min, PCR

activation at 95˚C for 5 min, denaturation at 95˚C for 15 sec, annealing at 60˚C during

60 sec and extension at 65˚C for 60 sec (Table 15). All amplification steps were

performed on a real-time thermo cycler, MiniOpticon System (Bio-rad Laboratories).

The denaturation, annealing and extention conditions were repeated for 45 cycles. To

minimize potential contamination mastermix and preparation the addition of the

samples were performed in separate rooms. In all the assays a positive control

(mengovirus RNA stock solution) and a negative control (RNase free water) were

included. The negative control were prepared at the same room as mastermix.

Table 15: Thermal conditions for detection of mengovirus by Qiagen qRT-PCR.





Annealing 60 sec/60ºC


37

3.5.2 Evaluation of viral mengovirus RNA extraction efficiency

The efficiency of extraction was determined for each sample, by comparing its Ct value

with the Ct value obtained with mengovirus stock solution (positive control) in the same

real-time RT-PCR assay and the difference (ΔCt) was used to calculate the percentage

by using the formula 100e-0.6978ΔCt

(Le Guyader et al., 2009).

38

4. RESULTS AND DISCUSSION

39

4. Results and Discussion

4.1 Detection of HEV RNA in shellfish by Qiagen qRT-PCR

4.1.1 Detection limit of the Qiagen qRT-PCR

The detection limit of Qiagen qRT-PCR was established using a 10 fold serial dilution

of the HEV stock solution (concentration of 108 RNA copies/μl), ranging from 1x10

9 to

1x100 copies/reaction. The number of HEV RNA copies and the correspondent Ct

values of each dilution are shown in Table 16. The correspondent amplification curves

of the dilution series can be observed in Figure 5 A.

Table 16: Ct values and number of copies of HEV RNA by Qiagen qRT-PCR.

HEV RNA copies/µl

(Stock solution)

HEV RNA copies/ copies/reaction (25µl)

HEV RNA copies/μl

(25µl)

Ct

1x108 1x10

9 4x10

7 10.16

1x107 1x10

8 4x10

6 12.79

1x106 1x10

7 4x10

5 16.36

1x105 1x10

6 4x10

4 20.05

1x104 1x10

5 4x10

3 23.2

1x103 1x10

4 4x10

2 27.37

1x102 1x10

3 4x10

1 30.87

1x101 1x10

2 4x10

0 34.11

1x100 1x10

1 4x10

-1 36.27

1x10-1

1x100 4x10

-2 33.62

Ct = Threshold cycle. Ten µl of each dilution, obtained from stock solution, in a total volume of 25µl was

tested.

As observed in table 16 and Figure 5 A the Ct value 33.62 of the concentration of 1

copy/reaction is lower than the one expected for a number of copies smaller than the 10

copies/reaction that showed a Ct of 36.27. This indicates that we are at the limit of the

detection of the assay.

The standard curve, that was performed in order to verify the efficiency of the

amplification of Qiagen qRT-PCR, was constructed without the value of the

concentration 1 copy/reaction for the present reason (Figure 5 B). An amplification

efficiency of 96% was obtained which is between the acceptable ranges of 90%-110%.

Based on all this results the limit of detection established for the Qiagen qRT-PCR was

10 copies/reaction HEV RNA, corresponding to the Ct of 36.27.

40

Hence for Qiagen qRT-PCR samples were considered:

i) Positive when they presented a Ct value < 36.27 (Ct value of the limit of

detection) associated to an exponential amplification curve;

ii) Negative when no amplification was observed or a Ct value was associated to

a non-exponential amplification curve;

iii) Above limit of detection (above LD), when sample Ct value was > 36.27 and

were associated to exponential amplification curve. These samples were interpreted

with caution because this high Ct value could be due to the presence of very low

concentrations of HEV RNA.

Figure 5: Establishment of the Qiagen qRT-PCR Standard curve for HEV. Standard curve was generated

using a 10 fold dilution of a HEV RNA stock solution, ranging from 1x109 to 1x10

0 copies/ tube (25μl).

A) Amplification curves of the dilution series (RFU- Relative fluorescence units). B) Standard curve with

the Ct plotted against the log of the starting quantity of template for each dilution. The equation for the

regression line is shown above the graph. The amplification efficiency was 96%.

A

B

41

4.1.2 HEV RNA detection in shellfish by Qiagen qRT-PCR

All the 81 RNA extracts from shellfish were tested for the presence of HEV RNA using

Qiagen qRT-PCR. Two different experiments performed in different days. The results

of these experiments are summarized in table 17. Samples were tested undiluted and

diluted 1:10. Dilution of shellfish samples is recommended since if there is presence of

derivatives inhibitors from extraction procedure and purification step nucleic acid

(guanidine or ethanol salts, for example), these inhibitors might affect the results,

producing late amplification or no amplification at all. Also, the low quantity of virus

in shellfish renders them a difficult and variable matrix that is also known to cause

amplification inhibition (Lowther et al., 2008). Some substances present in shellfish

tissue, like polysaccharides and glycogen (Atmar et al., 1993) can affect the removal of

the virus and its subsequent concentration, extraction of virus nucleic acids, and/or

inhibit nucleic acid amplification.

From the total of 81 shellfish samples tested, 20 were considered positive, 47 were

negative and 13 above LD. Concerning the positive samples B4, B38 and B53, they

gave only a positive result when tested undiluted, showing no amplification when tested

diluted. This could be due to a small quantity of HEV RNA that could not be detected

after the dilution of sample (less than 10 copies of HEV RNA/tube).

Samples B8, B21, B37, B43 and B59 showed Ct values above the limit of detection

(>36.27 ) when tested undiluted but no amplification when tested diluted. These

samples were not considered positive since we can not guarantee that their high Ct

values were due to the presence of a very small quantity of HEV RNA (not

detectectable the diluted sample) or due to a nonspecific reaction.

Samples B22 and B58 presented only a positive Ct value when tested diluted, and no

amplification without dilution. The absence of amplification when tested undiluted

could be explained by the presence of inhibitors. The presence of inhibition could also

explain the high Ct values observed with the diluted samples B51 (12.08) and B54

(18.41) when compared with the Ct of the diluted samples, 26.56 and 39.58,

respectively. The contrary would be expected since the undiluted samples would have

more HEV RNA and consequently a lower Ct value.

42

Table 17: Ct values and interpretation of results of undiluted and diluted shellfish

samples for the detection of HEV RNA by qiagen qRT-PCR. Samples 1st Experiment 2 nd Experiment Interpretation

Undiluted Diluted 1:10 Undiluted Diluted 1:10

Ct Ct B1 N/A N/A N/A N/A Negative

B2 N/A N/A N/A N/A Negative

B3 N/A N/A - - Negative

B4 30.34 N/A - - Positive


B6 N/A N/A N/A - Negative


B8 43.99 N/A - - Above LD

B9 N/A 40.34 - - Above LD

B10 N/A 41.58 - - Above LD

B11 4.77 25.94 - - Positive




B15 N/A 5.87 N/A N/A Negative





B20 16.75 - - 25.98 Positive

B21 45.48 N/A - - Above LD

B22 N/A 32.52 N/A N/A Positive


B24 21.97 34.19 - - Positive


B26 39.85 - 31.04 - Positive



B29 N/A N/A N/A N/A Negative




B33 N/A 38.79 - - Above LD




B37 43.44 N/A N/A - Above LD


B39 24.61 30.28 - - Positive




B43 37.63 N/A N/A - Above LD








B51 26.56 12.08 - - Positive

B52 N/A 43.46 - - Above LD


B54 39.58 18.41 - - Positive

B55 17.53 34.63 - 42.64 Positive

B56 7.98 6.82 - - Negative

B57 N/A 4.18 - - Negative

B58 N/A 24.94 - - Positive

B59 44.37 N/A - - Above LD

B60 7.96 9.07 - - Negative

B61 31.81 - - - Positive

B62 29.62 29.93 - - Positive

B63 42.04 40.36 N/A - Above LD

B64 4.42 1.63 - N/A Negative

B65 30.99 - - - Positive

B66 42.36 42.14 - - Above LD

B67 30.89 - - - Positive

B68 44.02 - N/A - Above LD

B69 38.99 45.49 - - Above LD

B70 13.58 26.20 - - Positive




B74 34.01 33.11 - - Positive

B75 4.01 3.86 N/A N/A Negative





B80 25.10 37.14 - - Positive

Ct=Threshold cycle, N/A=no amplification; LD= limit of detection

i) positive when they presented a Ct value lower than the limit of detection (36.27); ii) negative when no amplification was observed

or presented a very low Ct (<10) value associated to a non-exponential amplification; iii) above limit of detection (above LD) when their Ct value was > 36.27..

43

4.2 Detection of HEV RNA in shellfish by Kapa qRT-PCR

4.2.1 Detection limit of Kapa qRT-PCR

The detection limit of Kapa qRT-PCR was established using a 10 fold serial dilution of

the HEV stock solution (concentration of 108 RNA copies/μl), ranging from 5x10

8 to

5x10-1

copies/reaction. The number of HEV RNA copies and the correspondent Ct

values of each dilution are shown in Table 18. The correspondent amplification curve of

the dilution series can be observed in Figure 6 A.

Table 18: Ct values and number of copies of HEV RNA by Kapa qRT-PCR.

Ct= Threshold cycle; N/A= no amplification. Five µl of each dilution in a total volume of 20µl was tested.

As observed in table 18 and Figure 6 A the Ct value 35.43 of the concentration of 5x102

copy/reaction indicates that we are at the limit of the detection of the assay.

The standard curve was performed in order to verify the efficiency of the amplification

of Kapa RT-PCR (Figure 6 B). For the construction of the standard curve the

concentrations of 5x101, 5x10

0 and 5x10

-1 copies/µl that presented no amplification

were not included. An amplification of 86% efficiency was obtained which is out of the

acceptable range of 90%-110%.

The limit of detection established for the Kapa qRT-PCR was 500 copies/reaction of

HEV RNA, corresponding to the Ct of 35.43. That is much lower than the number of

copies the Qiagen qRT-PCR can detect (10 copies HEV RNA). This could be in part

explained by lower amplification efficiency presented of Kapa qRT-PCR (86%).

Hence for Kapa qRT-PCR samples were considered:

HEV RNA copies/µl

(Stock solution)

HEV RNA

copies/reaction (20µl)

HEV RNA

copies/μl (20µl)

Ct

1x108

5x108

2,5x107

12.73

1x107

5x107

2,5x106

15.11

1x106

5x106

2,5x105

18.76

1x105

5x105

2,5x104

21.86

1x104

5x104

2,5x103

25.26

1x103

5x103

2,5x102

29.65

1x102

5x102

2,5x101

35.43

1x101

5x101

2,5x100

N/A

1x100

5x100

2,5x10-1

N/A

1x10-1

5x10-1

2,5x10-2

N/A

44

i) Positive when they presented a Ct value < 35.43 (Ct value of the limit of detection);

ii) Negative when no amplification was observed;

iii) Above limit of detection (above LD) when their Ct value was >35.43. These

samples were interpreted with caution because this high Ct value could be due to the

presence of very low concentrations of HEV RNA.

Comparing the limit of detection of Qiagen and Kapa qRT-PCR, the Qiagen presented

higher sensitivity, with a limit of detection of 10 copies HEV RNA/µl versus Kapa

qRT-PCR, which showed a limit of detection of 500 copies HEV RNA/µl.

Figure 6: Establishment of the Kapa qRT-PCR standard curve for HEV. A standard curve was generated

using a 10 fold dilution of a HEV RNA solution ranging from 5x108 to 5x10

-1 copies/μl. A) Amplification

curves of the dilution series (RFU- Relative fluorescence units). B) Standard curve with the Ct plotted

against the log of the starting quantity of template for each dilution. The equation for the regression line

and the R2 value are shown above the graph. The amplification efficiency was 86%.

A

B

45

4.2.2 HEV RNA detection in shellfish by Kapa qRT-PCR

Only 22 shellfish samples were tested by Kapa qRT-PCR. They were all tested

undiluted. The interpretation of results obtained with Kapa qRT-PCR are summarized in

Table 19.

Table 19: Ct values and interpretation of undiluted shellfish samples for the detection

of HEV RNA by Kapa probe qRT-PCR.

Samples Undiluted

Ct

Interpretation

B8 33.40 Positive

B9 37.02 Above LD

B20 35.08 Positive

B22 N/A Negative

B23 N/A Negative

B33 33.38 Positive

B34 N/A Negative

B52 34.53 Positive

B55 30.09 Positive

B57 N/A Negative

B58 34.95 Positive

B59 39.43 Above LD

B61 33.18 Positive

B62 30.26 Positive

B63 37.27 Above LD

B64 N/A Negative

B65 33.01 Positive

B66 36.30 Above LD

B67 31.52 Positive

B68 39.93 Above LD

B69 34.96 Positive

B70 25.98 Positive

Ct=Threshold cylce; N/A= no amplification, LD= limit of detection

i) positive when they presented a Ct value lower than the limit of detection (35.43); ii) negative when no

amplification was observed iii) above limit of detection (above LD) when their Ct value was >35.43.

From the total of 22 shellfish samples tested, 12 (54.5%) were positive, 5 (4.54%) were

negative and 5 (4.54%) were above LD.

46

When we compare the final results of the 22 samples obtained by Qiagen and Kapa

qRT-PCR (Table 20) it can be observed that only 8 samples were positive by both

assays namely B20, B55, B58, BB61, B62, B65, B67, B69 and B70. Samples B8, B33,

B52 and B69, presented Ct values above the limit of detection when tested by Qiagen

qRT-PCR, but were positive using Kapa qRT-PCR. This shows how important it is to

be caution with the interpretation of above LD samples. The high Ct value of sample B8

that led to an interpretation of “above LD” was really due to the presence of low

quantity of copies of HEV RNA.

Table 20: Comparison of the interpretation of results of 22 shellfish samples tested by

both Qiagen and Kapa qRT-PCR.

Samples Interpretation* Interpretation

B8 Above LD

Positive

B9 Above LD Above LD

B20 Positive Positive

B22 Positive Negative

B23 Negative Negative

B33 Above LD Positive

















*These results were interpreted after considering also the Ct with the samples diluted 1:10. Ct= Threshold cycle,

N/A= no amplification; LD= limit of detection; N/C= not conclusive.

47

4.3 Detection of HEV RNA in shellfish by Kapa Sybr® qPCR

4.3.1 Kapa Sybr® qPCR Optimization

A previous optimization of this Kapa Sybr® qPCR was performed. Two different

annealing-extension temperature 60ºC and 64ºC were used. As can be observed the

increase of the annealing temperature from 60ºC (Figure 7 A) to 64ºC (Figure 7 B) led

to the disappearance of the formation of primer dimers responsible for the amplification

curve detected in the negative control.

Hence the best annealing temperature was 64ºC and all the experiments of detection of

HEV RNA in shellfish used this temperature.

Figure 7: Optimization for HEV detection by Kapa Sybr® qPCR. Amplification and melting curves

analysis. Amplification plot curve of HEV ten-fold serial dilutions (107to 10

3 RNA copies) of HEV RNA

stock solution, using primers concentration of 300nM and annealing temperature of 60ºC (A) and 64ºC

(B); Melting curve with annealing temperature of 64ºC (C).

A

B

48

The figure 8 is represented the melting curves of the serial dilutions with annealing

temperature of 64ºC.

Although the primers proposed by Jothikumar et al., (2006) were designed to work with

probes, these primers showed to work well also with Sybr Green, as demonstrated by

the melting curve (Figure 8). All amplified products presented the same expected

melting temperature of 79 º C.

Figure 8: Melting curves of the serial dilutions with annealing temperature of 64ºC. The melting curves

shows that the melting peak of the serial dilutions (107 to10

3) was at 79ºC.

The annealing of the primers to their target sequences is critical step in a PCR reaction.

It has to be performed at the right temperature for the primers to anneal efficiently to

their targets, while preventing nonspecific annealing and primer-dimer formation

(Taylor et al., 2011). Optimization of reaction conditions can reduce primer-dimer

formation and increase the efficiency and specificity of the amplification process

(Raymaekers et al., 2009).

49

4.3.2 Detection limit of Kapa Sybr® qPCR

The detection limit of Kapa Sybr® qPCR was established using a 10 fold serial dilution

of the HEV stock solution (concentration of 108 RNA copies/μl), ranging from 5x10

5 to

5x100

copies/reaction. The number of HEV RNA copies and the correspondent Ct

values of each dilution are shown in Table 21. The correspondent amplification curves

of the dilution series are in Figure 9 A.

Table 21: Ct values and melting curve of HEV RNA stock solution by Kapa Sybr®

qPCR.

HEV RNA

copies/μl (Stock

solution)

HEV RNA

copies/reaction

(20μl)

HEV RNA

copies/μl

(20μl)

Ct Melting curve

temperatre

1x105 5x10

5 2,5x10

4 21.27 79,00

1x104 5x10

4 2,5x10

3 24.54 79,00

1x103 5x10

3 2,5x10

2 28.19 79,00

1x102 5x10

2 2,5x10

1 31.10 78,80

1x101 5x10

1 2,5x10

0 35.49 78.80

1x100 5x10

0 2,5x10

-1 37.44 78,60

Ct= Threshold cycle; N/A= no amplification. Five µl of each dilution in a total volume of 20µl was

tested.

A standard curve was also performed in order to verify the efficiency of the

amplification of Kapa Sybr® qPCR (Figure 9 B). For the construction of the standard

curve the concentration of all dilution series were included. An amplification efficiency

of 100% was obtained.

The limit of detection established for Kapa Sybr® qPCR was 5 copies of HEV

RNA/reaction corresponding to the Ct of 37.44.

Hence for Kapa Sybr® qPCR samples were considered:

i) Positive when they presented Ct < 37.44 and a melt temperature ranging

between 78,5º C - 79ºC.

ii) Negative when no amplification was observed or when an amplification was

present but with a melt temperature different from 78,5º C - 79ºC.

50

Figure 9: Amplification curves of standards stock solution Kapa Sybr® qPCR. A standard curve was

generated using a 10 fold dilution of a HEV RNA stock solution ranging from 5x105 to 5x10

-1 copies/μl).

A) Amplification curves of the dilution series. B) Standard curve with the Ct plotted against the log of the

starting quantity of template for each dilution. The calculated amplification efficiency was 100%; R2:

0,993 and slope: -3.309.

A

B

51

4.3.3 HEV RNA detection in shellfish by Kapa Sybr® qPCR

A total of 33 extracts from shellfish were tested undiluted and diluted (1:5), for the

presence of HEV. The Ct values, the melt temperature and the interpretation of results

shellfish samples are summarized in Table 22.

Table 22: Ct values, melt temperatures and interpretation of results of undiluted and

diluted 1:5 shellfish samples by Kapa Sybr® qPCR for the detection of HEV RNA.

Samples

Undiluted

Diluted 1:5

Ct Melt temperature (ºC) Result Ct Melt temperature (ºC) Result B8 N/A None Negative 35.26 80,40 Negative

B9 N/A None Negative 36.05 78,80 Positive

B10 N/A None Negative 34.65 None Negative


B20 N/A None Negative 37.32 72,40 Negative









B51 N/A None Negative N/A None Negative






B59 N/A None Negative 36.11 72.40 Negative












B80 38.09 78,60 Positive 38.20 72,60 Negative

Ct=Threshold cycle; N/A= no amplification. Positive: when they presented Ct < 37.44 and a melt temperature

ranging between 78,5º C to 79ºC. Negative when no amplification was observed or even with amplification but with

melt temperature different from 78,5º C to 79ºC.

52

From the 33 shellfish samples tested undiluted, only one sample (B80) was considered

to be positive. Althouhg with a late amplification (Figure 10 A) its Ct value (38.09)

was above of the limit of detection (37.44), observing the melt curve is possible to see

that the sample B80 had the expected melting temperature (78,6ºC) of the HEV RNA

control (Figure 10 B).

A

B

Figure 10: Amplification curves of shellfish samples tested undiluted by Kapa Sybr® qPCR. A)

Amplification curves of the dilution series and sample B80 the single one that amplified. B) Melt

curve showing the melt temperatures of serial dilutions and the sample B80.

53

From the 33 shellfish samples tested diluted, 10 samples (30,3%) were considered

positive namely B9, B11, B58, B61, B63, B67, B80 since these samples presented a Ct

< 37.44. Samples B43 (Ct=37.60), B55 (Ct=38.74) and B68 (Ct=38.13), were also

considered positive. Although their Ct of samples were slightly above the Ct limit of

detection (37.44) and melt temperature were around 79 ºC (a difference of 0,5ºC in the

melting temperature was acceptable.

Analyzing the Ct values of the diluted samples it can be observed that almost all showed

amplification (Table 22). However the melting temperatures of the majority of samples

are different from the expected 79ºC correspondent to the specific amplified products.

The presence of inhibitors in undiluted samples was also observed in this assay. In fact

the samples B8, B9, B11, B43, B55, B61, B68, and B69 show no amplification when

tested undiluted but show a specific amplification when tested diluted.

The figure 11, shows the amplification curves (A) and melt temperature curves (B) of

the shellfish samples tested diluted that were considered positive (B8, B9, B11, B43,

B55, B61, B68, and B69) since they all exhibit a melting temperatures around 79ºC.

54

A

B

Figure 11: Amplification plot of samples tested diluted by Kapa Sybr® qPCR that were

considered positive. A) Amplification curves of the dilution series and shellfish samples

considered positive (B9, B11, B43, B55, B58, B61, B63, B67, B68 and B80). B) Melting

curve showing the melting temperature of the HEV RNA dilutions and the samples

considered positives.

55

4.4 Detection of HEV RNA by nested RT-PCR

A total of 33 potential positive samples, based on the results of real-time RT-PCR, were

tested by conventional nested RT-PCR. This will allow the confirmation of presence of

HEV RNA and by sequences of the expected amplified products product (330 bp).

Samples were also tested undiluted and diluted (1:5 and 1:10) in order to overcome the

presence of inhibitors. The results are presented in Table 23.

Table 23: Results of the detection of HEV RNA in shellfish samples by nested RT-PCR

Sample Undiluted Diluted 1:5 Diluted 1:10

B8 Negative Positive Negative

B9 Negative Negative Negative

























B66 Positive Negative Negative







When samples were tested undiluted, only sample B66 presented the expected amplicon

of 330bp (Figure 11 A), but when tested diluted (1:5) five samples namely B8, B10,

B11, B34 and B55 (Figure 11 B and C) showed also the 330bp band. These results

show once again how important is to test the samples diluted to avoid underestimation

of virus positive samples due to the presence of inhibitors. However samples must be

56

tested undiluted to avoid underestimation of samples with low viral concentration.

Sample B66 is a good example of sample that would be missed if it was only tested

undiluted.

A - Shellfish samples tested undiluted

B - Shellfish samples tested diluted (1:5)

C - Shellfish samples tested diluted (1:5)

Figure 12: Electrophoresis gel of amplified products after nested RT-PCR shellfish samples. A)

Samples tested undiluted. B66 show the expected amplicon of 330bp. B) and C) Samples tested

diluted (1:5) samples (B8, B10, B11, B34 and B55) show the expected amplicon of 330bp. C+:

positive control.

57

A final comparison of the results obtained by nested RT-PCR and real-time RT-PCR

were made (Table 24). It can be observed that only sample B11 and B55 were positive

in all the assays tested. Some samples like B34, B8, B10, B66 that were negative or

above the limit of detection by real-time RT-PCR assays were positive with nested RT-

PCR. This could be explained by the presence of low HEV RNA concentration in the

samples. On the contrary the samples B20, B58, B65, B67, B70 and 80 that were

positive in at least two real-time RT-PCR assays were negative with nested RT-PCR.

The presence of HEV RNA in these samples must be interpreted with caution since the

primers used for real time assays and nested RT-PCR target a different region of the

ORF HEV genome.

Table 24: Comparison of the results by real-time RT-PCR and nested RT-PCR assays

in 33 shellfish samples.

Samples

Real-time RT-PCR

Nested RT-PCR

Qiagen qRT-PCR Kapa qRT-

PCR

Sybr qRT-PCR

B8 Above LD Positive Negative Positive

B9 Above LD Above LD Positive Negative

B10 Above LD - Negative Positive

B11 Positive - Positive Positive

B20 Positive Positive Negative Negative

B21 Above LD - Negative Negative

B23 Negative Negative Negative Negative

B24 Positive - Negative Negative

B33 Above LD Positive Negative Negative

B34 Negative Negative Negative Positive



B43 Above LD - Positive Negative


B52 Above LD Positive Negative Negative



B55 Positive Positive Positive Positive

B58 Positive Positive Positive Negative

B59 Above LD Above LD Negative Negative

B60 Negative - Negative Negative

B61 Above LD Positive Positive Negative



B64 Negative Negative Negative Negative


B66 Above LD Above LD Negative Positive*

B67 Positive Positive Positive Negative


B69 Above LD Positive Positive Negative



B80 Positive - Positive* Negative

*This sample was positive only when was tested undiluted.

58

4.5 Sequencing of HEV nested RT-PCR products

The amplified products of 330 bp obtained from samples B8, B10, B11, B34, B55 and

B66, were sequenced and further compared with known HEV reference strains in order

to evaluate their genetic relationship.

The phylogenetic analysis of the HEV products demonstrated that all strains belonged

to HEV genotype 3 subgenotype e (Figure 13), and that they are closely related to

human strains reinforcing the potential risk to public health.

Figure 13: Phylogenetic tree based on the nucleotide sequence of the ORF1. The HEV strains of this

study are named Shellfish 8, 10, 11, 34, 55 and 66. HEV sequences were obtained from GenBank.

59

4.6 Evaluation of viral RNA extraction efficiency

The extraction efficiency of the nucleic acid of all the shellfish samples was evaluated.

For that samples were previously spiked with mengovirus before being processed. The

comparison of the mengo spiking concentration with the concentration of mengo found

after extraction, defines the acceptability of the recovery efficiency of the extraction

process.

The efficiency extraction was calculated for 23 shellfish samples. That were tested

undiluted and diluted (1:10) by Qiagen qRT-PCR for the presence of mengovirus RNA.

The extraction efficiency was calculated using the difference between the Ct value of

the sample and the Ct value of the mengovirus used as positive control in the assay. The

values of the extraction efficiency of the 23 samples are showed in Table 25 and the

amplification curves in Figure 13.

The extraction efficiency of the majority of shellfish samples presented efficiency

>40%, which indicates that mengovirus was successfully recovered from the molluscan

shellfish. Being expected that HEV has been also successfully extracted. For samples

that present an extraction efficiency <10% it is recommended to perform a new

extraction.

60

Table 25: Extraction efficiency of undiluted and diluted shellfish samples spiked with

mengovirus. Samples CtaMengovirus Cta samples ΔCt/3,3b %

Extractionc B1 32.26 34.39 0.645 63.74

B1 1:10 32.26 36.06 1.151 44.77

B2 32.26 33.72 0.442 73.44

B2 1:10 32.26 32.74 0,145 90.35

B3 32.26 27.06 -1.575 300.29

B3 1:10 32.26 33.17 0.275 82.50

B4 32.26 34.73 0.748 59.32

B4 1:10 32.26 34.7 0.739 59.69

B5 32.26 33.32 0.321 79.92

B5 1:10 32.26 34.83 0.778 58.07

B6 32.26 34.24 0.6 65.79

B6 1:10 32.26 33.96 0.515 69.80

B7 32.26 32.71 0.136 90.92

B7 1:10 32.26 34.03 0.536 68.78

B8 32.26 33.9 0.496 70.70

B8 1:10 32.26 33.68 0.248 84.08

B9 32.26 33.15 0.269 82.85

B9 1:10 32.26 37.24 1.509 34.89

B10 32.26 34.49 0.675 62.40

B10 1:10 32.26 35.81 1.075 47.21

B11 32.26 37.56 1.606 32.60

B11 1:10 32.26 39.34 2.145 22.38

B12 32.26 36.92 1.412 37.33

B12 1:10 32.26 35.2 0.890 53.70

B13 32.26 35.06 0.848 55.32

B13 1:10 32.26 37.06 1.454 36.24

B14 32.26 33.03 0.233 84.97

B14 1:10 32.26 33.82 0.472 71.90

B15 32.26 36.64 1.327 39.60

B15 1:10 32.26 34.47 0.669 62.66

B16 32.26 33.68 0.430 74.06

B16 1:10 32.26 35.65 1.027 48.82

B17 32.26 36.92 1.412 37.32

B17 1:10 32.26 36.77 1.366 38.53

B18 32.26 34.31 0.621 64.82

B18 1:10 32.26 38.35 1.845 27.58

B19 32.26 35.76 1.060 47.70

B19 1:10 32.26 35.16 0.878 54.16

B20 32.26 35.23 0.9 53.36

B20 1:10 32.26 13.3 -5.745 5510.15

B21 32.26 30.61 -0.5 141.75

B21 1:10 32.26 17.59 -4.445 2224.32

B22 32.26 33.74 0.448 73.12

B22 1:10 32.26 32.43 0.051 96.46

B23 32.26 5.64 -8.066 27836.13

B23 1:10 32.26 34.7 0.739 59.69

aCt=threshold cycle;

b Slope;

cExtraction efficiency was calculated using 100

e-0.6978ΔCt(ΔCt=Ct sample-Ct mengovirus;

e=exponencial).

61

Some samples showed extraction efficiencies >100%, which means that more

“mengovirus RNA” is being detected than those seeded in the sample, what is

impossible. For example, sample B23 (undiluted) presents an aberrant value 27836.13%

that is a consequence of its Ct value of 5.64 (Figure 5). However when B23 was tested

diluted an acceptable value of efficiency was obtained 59.69%. Matrix interference

could be behind this type of aberrant value observed in undiluted samples but this

problem are normally solved by testing samples after dilution, as observed with B23.

Figure 14: Mengovirus amplification curves of undiluted and diluted shellfish samples by real-Time RT-

PCR. The amplification curve of sample B23 (undiluted) shows an early amplification which shape

suggests a possible interference of the matrix.

The addition of an external virus like mengovirus to a shellfish sample has been

proposed as a control to evaluate the extraction efficiency of molecular virus detection

methods (Costafreda et al., 2006). Mengovirus, which was used in the presented study,

has been considered a good candidate for that purpose by the fact that this virus is

unlikely to naturally contaminate shellfish, it is non-pathogenic for humans and is very

similar to enteric viruses, to be a non-enveloped virus with a single strand RNA (Le

Guyader et al., 2009).

62

5. CONCLUSIONS

63

5. CONCLUSIONS

The results of the present project lead to the follow conclusions:

1-The three in house real-time RT-PCR assays used to detect HEV RNA in shellfish

samples, targeting a conserved region within the ORF 3 of the HEV genome, showed

different limits of detection, namely:

10 copies of HEV RNA/reaction for the Qiagen qRT-PCR (probe)

corresponding to a Ct value of 36.27.

500 copies of HEV RNA/reaction for the Kapa qRT-PCR (probe) corresponding

to a Ct value of 35.43.

5 copies of HEV RNA/reaction for Kapa Sybr® qPCR (Sybr Green)

corresponding to a Ct value of 37.44.

2- The results of the detection of HEV RNA in the shellfish samples with these three in

house real-time RT-PCR assays were:

19 samples from 81 tested were positive by Qiagen qRT-PCR;

12 samples from 22 tested were positive by Kapa qRT-PCR;

10 samples from 33 tested were positive by Sybr qRT-PCR.

Only 3 samples were found positive from the total of 20 that were tested by all the

three in house real-time RT-PCR assays. However if samples classified as “above

LD” were also considered (because their high Ct values could be due to the presence

of very low concentrations of HEV RNA) the number of positive samples increase

to 10.

3- From the shellfish samples positive for HEV RNA by real-time RT-PCR 6 were

positive when tested with nested RT-PCR targeting the ORF1 of the HEV genome. The

amplified products of 330bp obtained were sequenced and the phylogenetic analysis

demonstrated that they belonged all to HEV genotype 3 subgenotype e.

4- The results from the RT-PCR assays (either real time or nested) showed how

important is to test the shellfish samples diluted to avoid underestimation of virus

positive samples due to the presence of inhibitors. Similarly, samples must be also

64

tested undiluted to avoid underestimation of samples with low viral concentration. In

fact, the detection of viruses in shellfish is mainly hampered by the presence of

inhibitors of the PCR and low viral concentration.

From our knowledge this is the first study that describe the presence of HEV

genotype 3 in shellfish cultivated in coastal area of Iberian Peninsula although the

presence of other human enteropathogenic viruses, like norovirus, hepatitis A virus and

enterovirus, has already been reported (Romalde et al., 2002; Mesquita et al., 2011;

Manso et al., 2013). This indicates that HEV is contaminating and circulating in the

estuary water environment around those shellfish beds. Whether shellfish were exposed

to human or animal fecal-polluted sewage needs to be further investigated. Other

previous studies have detected the presence of a swine-like HEV genotype 3 in bivalve

mollusks (Li et al., 2007; Crossan et al., 2012; Gao et al., 2015), suggesting the

presence of HEV contamination in the coastal waters where those shellfish were

cultivated.

Bivalve shellfish beds are at constant risk of being exposed to contamination as a

consequence of runoff waste from sewage treatment plants or slurry fertilized fields

(Krog et al., 2014). It is possible that the number of HEV particles discharged into the

environment is too low to detect or that the virus may have a very short period of

persistence in pig manure and human waste (Grodzki et al., 2014) hampered these

investigations.

There are not many studies about the impact of HEV in shellfish although there

are findings suggesting that a health risk may exist for shellfish consumers (Gao et al.,

2015). The evaluation of the human health risk of consuming shellfish from areas where

infectious HEV is present is mandatory.

65

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66

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